BR112013018898B1 - USE OF GLICYL-L-2-METHYLPROLYL-L-GLUTAMIC ACID AND ANALOGUES FOR THE TREATMENT OF AUTISM SPECTRUM DISORDERS - Google Patents

Abstract

TREATMENT OF AUTISM SPECTRUM DISORDERS USING GLYCYL-L-2-METHYLPROLYL-L-GLUTAMIC ACID This invention provides compounds, compositions and methods for treating autism spectrum disorders (ASD) employing 2-glycyl-2-methylpropyl-glutamic acid (G-2-MePE) and analogues thereof. Autism spectrum disorders include autism, autism disorder, Asperger syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS), fragile X chromosome syndrome, and Rett syndrome. Compositions containing compounds include water-soluble formulations, water-in-oil microemulsions, coarse water-in-oil emulsions, water-in-oil liquid crystals, nanocapsules, tablets and orally administered gels. The compounds and compositions of this invention can be administered intravenously, intraventicularly or orally and can be effective in treating neurodegeneration, promoting neurological function, treating crisis activity in diseases and other symptoms of ASD and can prolong life in animals including human beings. humans who have autism spectrum disorders.

Description

Priority Claim

[001] This application claims priority to Provisional US Patent Application Number 61/462,141 entitled "Rett Syndrome Therapy Using Glycyl-2-Methylpropyl-L-Glutamate", Larry Glass et al., Inventors, filed January 27 of 2011, and Provisional US Patent Application number 61/492,248 entitled "Treatment of Autism Spectrum Disorders Using Glycyl-2-L-Methylprolyl-L-Glutamate", Michael John Bickerdike and other inventors, filed June 1, 2011. Both orders are incorporated into this order in their entirety as if incorporated separately.

Field of Invention

[002] This invention relates generally to the treatment of autism spectrum disorders (ASD), including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autism Disorder, Asperger Syndrome, Childhood Disintegrative Disorder, and Disorder Developmental Pervasive Not Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA). In particular, this invention relates to the treatment of ASD employing Glycyl-2-Methyl-Prolyl-Glutamate (G-2-MePE).

BACKGROUND Description of Related Technique

[003] Autism spectrum disorders are becoming increasingly diagnosed. Autism spectrum disorders (ASD) are a set of linked developmental disorders characterized by abnormalities in social interaction and communication, restricted interests, and related behaviors. The current classification of ASD recognizes five distinct forms: classic autism or autism disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder, and pervasive developmental disorders not otherwise specified (PDD-NOS). The sixth syndrome, pathological demand avoidance (PDA), is yet another pervasive developmental disorder.

[004] EP 0 366 638 discloses GPE (a tri-peptide consisting of amino acids Gli-Pro-Glu) and its di-peptide derivatives Gli-Pro and Pro-Glu. EP 0 366 638 reveals that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

[005] WO95/172904 describes that GPE has neuroprotective properties and that the administration of GPE can reduce damage to the central nervous system (CNS) by preventing or inhibiting the death of neuronal and glial cells.

[006] WO 98/14202 describes that the administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system ( CNS).

[007] WO99/65509 describes that by increasing the effective amount of GPE in the CNS, such as by administering GPE, one can increase the effective amount of tyrosine hydroxylase (TH) in the central nervous system to increase dopamine-mediated production by TH in the treatment of diseases such as Parkinson's disease.

[008] WO02/16408 discloses certain analogues of GPE which have amino acid substitutions and certain other modification which are capable of inducing a physiological effect equivalent to GPE within a patient. Applications of GPE analogues include the treatment of acute brain injury and neurodegenerative diseases, including CNS injury or disease.

SUMMARY

[009] There is no current effective treatment for ASD and patient care is limited to symptom management.

[0010] This invention relates to synthetic and peptidomimetic analogues of glycyl-L-prolyl-L-glutamic acid (GPE). In particular, this invention relates to GPE analogs and peptidomimetics which are anti-apoptotics and anti-necrotics, to methods for preparing the same, to pharmaceutical compositions containing them, and to their use to improve cognitive function and/or treatment of memory disorders and to improve neuronal connectivity in animals. More specifically, this application refers to methods of using the analog GPE, G-2-methyl-prolyl-glutamic acid (G-2-MePE) in the treatment of autism.

[0011] The US Patent number. 7,041,314 describes compositions of matter and methods of using G-2-MePE.

em que m é 0 ou 1; n é 0 ou 1; X é H ou -NR6 R7; Y é H, alquila, - CO2R5, ou -CONR6 R7; Z é H, alquila, - CO2R5 ou -CONR6 R7; R1 é H, alquila ou aralquila; R2, R3, e R4 são, independentemente, H ou alquila; cada R5 é independentemente H, alquila, ou um resíduo de álcool graxo; cada R6 e R7 é independentemente H, alquila, ou aralquila, ou -NR6 R7 é pir- rolidina, piperidina, ou morfolina; ou uma lactona formada quando um composto em que Y é -CO2 (alquila) e Z é -CO2H ou onde Y é - CO2H e Z é -CO2 (alquila) é lactonizado; e os seus sais farmaceuticamente aceitáveis, desde que o composto não seja GPE, N-Me-GPE, amida de GPE, APE, GPQ ou um sal do mesmo.[0012] In one aspect, this invention provides compounds of formula 1 and formula 2:

where m is 0 or 1; n is 0 or 1; X is H or -NR6 R7; Y is H, alkyl, -CO2R5, or -CONR6 R7; Z is H, alkyl, -CO2R5 or -CONR6 R7; R1 is H, alkyl or aralkyl; R2, R3, and R4 are independently H or alkyl; each R5 is independently H, alkyl, or a fatty alcohol residue; each R6 and R7 is independently H, alkyl, or aralkyl, or -NR6 R7 is pyrrolidine, piperidine, or morpholine; or a lactone formed when a compound where Y is -CO2 (alkyl) and Z is -CO2H or where Y is -CO2H and Z is -CO2 (alkyl) is lactonized; and pharmaceutically acceptable salts thereof, provided the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt thereof.

[0013] Another aspect of the invention provides methods for treating an animal with an autism spectrum disorder comprising administering an effective amount of Glycyl-L-2-methylprolyl-L-glutamic acid (G-2-MePE) to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] This invention is described with reference to specific embodiments thereof. Other aspects and characteristics of the present invention can be understood with reference to the figures, in which:

[0015] Figure 1 is a general scheme for the preparation of synthetic GPE analogs of the invention.

[0016] Figures 2 and 3 show schemes for modifying glycine residues in GPE.

[0017] Figures 4 to 9 describe schemes for modifying glutamic acid residues of GPE.

[0018] Figures 10 and 11 illustrate schemes for modifying the peptide bonds of GPE.

[0019] Figures 12-15 show graphs summarizing the results of in vitro neuron testing with GPE or G-2-MePE and okadaic acid.

[0020] Figure 12 shows a graph illustrating the effects and GPE on cortical neurons injured with okadaic acid.

[0021] Figure 13 is a graph showing the effects of G-2-MePE on cortical neurons injured with okadaic acid.

Figure 14 is a graph showing the effects of G-2-MePE, GPE on cerebellar microexplants injured with okadaic acid.

[0023] Figure 15 is a graph showing the effects of G-2-MePE or GPE on striatal cells injured with okadaic acid.

[0024] Figure 16 shows the effects of subcutaneous injection of G-2-MePE (at doses of 0.012, 0.12, 1.2 and 12 mg/kg) on the number of ChAT-positive neurons in the striatum of rats 18 months of age.

[0025] Figure 17 shows the effects of G-2-MePE treatment on spatial memory retention in middle-aged 12-month-old rats.

Figures 18A and 18B show effects of G-2-MePE on spatial working memory of 17-month-old rats in an 8-arm radial maze after three weeks of treatment and nine days of resistance. Figure 18A shows the maze acquisition profiles across days for the different groups. Figure 18B shows the proportion of correct choices on average across days for the groups.

Figure 19A shows the effects of a single intraperitoneal administration of four doses of G-2-MePE on neuroblast proliferation as judged by the number of PCNA positive cells in the subventricular zone (SVZ) of aged rats.

Figure 19B shows the effects of a single intraperitoneal administration of four doses of G-2-MePE on the colocalization of PCNA and double-curtain staining in a rat treated with the highest dose of G -2-MePE (right panel) compared to vehicle treated rats (left panel).

Figure 19C shows the effects of G-2-MePE on the proliferation of PCNA neuroblasts as assessed by immunohistochemical staining in middle-aged rats.

Figure 20A shows a significant increase in the number of reactive astrocytes as assessed by GFAP staining in the hippocampus of elderly rats compared to young rats (*p<0.01) and middle aged rats (*p<0.01).

[0031] Figure 20B shows a photograph of a section of the cerebral cortex of an aged rat, showing astrocytes as assessed with GFAP staining, some of which are associated with capillary formation (arrows).

[0032] Figure 20C shows the dose-dependent effects of G-2-MePE treatment (at doses of 0.12, 0.12, 1.2 and 12 mg/kg/day) on astrocyte number reduction as assayed using GFAP staining in the CA4 subregion of the hippocampus in aged rats.

[0033] Figure 20D shows the dose-dependent effects of G-2-MePE treatment (at doses of 0.12, 0.12, 1.2 and 12 mg/kg/day) on astrocyte number reduction as assayed using GFAP staining in the cerebellar cortex.

[0034] Figure 21 shows the pharmacokinetic properties of GPE and G-2-MePE in the circulation of rats after intravenous injection.

Figure 22 shows the effects of G-2-MePE in increasing survival duration in MeCP2 deficient mice compared to MeCP2 deficient mice treated with brine.

[0036] Figure 23 shows the effects of G-2-MePE on long-term hippocampal potentiation as measured by the fEPSP slope in MeCP2 deficient mice compared to MeCP2 deficient mice treated with brine solution.

[0037] Figure 24 represents a graph showing the effects of G-2-MePE on dendrite length as a function of distance from the sum of cells.

DETAILED DESCRIPTION Definitions

[0038] The term "about", with reference to a dose or time refers to a certain variable and a range around that variable that is within the normal measurement error is 20% of the value of the variable. The term "about" with reference to an observed result means that the variation is within 20% of the value of the observed variable.

The term "alkyl" means a linear saturated hydrocarbon group having from one to six carbon atoms, or a saturated, branched or cyclic hydrocarbyl group having from three to six carbon atoms. Exemplary alkyl groups include straight or branched chain, or cyclic alkyl groups, methyl, ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, and hexyl.

The term "animal" includes humans and non-human animals, such as domestic animals (dogs, cats, and the like) and farm animals (cattle, horses, sheep, goats, pigs, and the like).

The term "aralkyl" means a group of the general formula -(CH2) 1-2Ar, where Ar is a 5- or 6-membered carbocyclic or heterocyclic aromatic ring, optionally substituted with 1 to 3 substituents selected from Cl, Br, -OH, -O-alkyl, -CO2R8 (where R8 is H or alkyl) or -NR8R9 where R8 is as described above and R9 is H or alkyl. Examples of aralkyl groups include benzyl, 2-chlorobenzyl, 4-(dimethylamino)benzyl, phenethyl, 1-pyrrolylmethyl, 2-thienylmethyl, 3- and pyridylmethyl.

The term "disease" includes any unhealthy condition of an animal, including in particular Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, diabetes, motor disorders, seizures, cognitive dysfunction due to aging and autism spectrum disorders, including autism, Fragile X Syndrome, Rett Syndrome (RTT), Autism Disorder, Asperger Syndrome, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), and Avoidance Demand Pathology (PDA).

[0043] The term "fatty alcohol residue" is a linear hydrocarbyl group having from seven to twenty carbon atoms, optionally containing up to three carbon-carbon double bonds. Exemplary fatty alcohol residues include decyl, pentadecyl, hexadecyl (cetyl), octadecyl (stearyl), oleyl, linoleyl, and eicosyl.

The term "growth factor" means an extracellular signaling polypeptide molecule that stimulates a cell to grow or proliferate.

The term "injury" includes any acute injury to an animal, including non-hemorrhagic stroke, traumatic brain injury, perinatal asphyxia associated with fetal distress, such as that following detachment, cord occlusion or associated with intrauterine growth retardation , perinatal asphyxia associated with inadequate resuscitation or breathing, severe CNS insults associated with near drowning, near death in the crib, inhalation of carbon monoxide, ammonia or other gaseous intoxication, cardiac arrest, coma, meningitis, hypoglycemia and state epileptic, episodes of cerebral asphyxia associated with coronary artery bypass graft surgery, episodes of hypotension and hypertensive crises, brain trauma and toxic injury.

[0046] "Memory disorders" or "cognitive disorders" are disorders characterized by permanent or temporary disability or loss of ability to learn, memorize or recall information. Memory disturbance can result from normal aging, brain damage, tumors, neurodegenerative diseases, vascular diseases, genetic diseases (Huntington's disease), hydrocephalus, other diseases (Pick's disease, Creutzfeld-Jakob disease, AIDS, meningitis), toxic substances, nutritional deficiency, biochemical disorders, psychological or psychiatric disorders. The presence of memory disorder in a human being can be established by in-depth analysis of the patient's history, physical examination, laboratory tests, imaging tests and neuropsychological tests. Standard neuropsychological tests include but are not limited to the Revised Brief Visual Memory Test (BVMT-R), Cambridge Automated Neuropsychological Testing Battery (CANTAB), Children's Memory Scale (CMS), Contextual Memory Test, Memory Test Recognition Test (CMRT), Controlled Oral Word Association Test and Memory Functioning Questionnaire, Denman Neuropsychology Memory Scale, Wechster Adult Intelligence Scale III Numerical and Alphabetical Sequence and Digit Subtest, Wechster Memory Assessment for Fuld Objects (HUNGER), Graham-Kendall Memory for Design Tests, Guild Memory Test, Hopkins Verbal Learning Test, Memory and Learning Testing Battery (LAMB), Clinical Self-Rating Memory Rating Scale (MAC-S), Memory Rating Scales (MAS), Randt Memory Test, Recognition Memory Test (RMT), Rey Auditory and Verbal Learning Test (RAVLT), Rivermead Behavioral Memory Test, Russell Adaptation Wechsler Memory Scale (RWMS), Spatial Working Memory, Memory and Learning Test (TOMAL), Vermont Memory Scale (VMS), Wechsler Memory Scale, Assessment Reach Memory and Learning - (WRAML).

The term "pharmaceutically acceptable excipient" means an excipient that is useful in the preparation of a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use . Such excipients can be solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous.

The term "pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable and has the desired pharmacological properties. These salts include salts that can be formed when acidic protons present in the compounds react with inorganic or organic bases. Suitable such inorganic salts include those formed with alkali metals, for example sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include those formed with organic bases such as amines, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Salts also include acid addition salts formed by reacting an amine group or groups present in the compound with an acid. Suitable acids include inorganic acids (eg hydrochloric and hydrobromic acids) and organic acids (eg acetic acid, citric acid, maleic acid, and alkane-and arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid) . When there are two acid groups present in a compound, a pharmaceutically acceptable salt may be a mono acid, mono salt or di-salt, and likewise, when there are more than two acid groups present, some or all of these groups may be salified. The same reasoning applies when two or more amine groups are present in a compound.

[0049] The term "protecting group" is a group that selectively blocks one or more reactive sites on a multifunctional compound, such that a chemical reaction can be selectively carried out on another unprotected reactive site and such that the group can be easily removed after the selective reaction is complete.

The term "therapeutically effective amount" means the amount of an agent that, when administered to an animal for treatment of a disease, is sufficient to effect treatment for the disease, as measured using an art-recognized test system .

[0051] The term "treating" or "treatment" of a disease may include preventing the disease from occurring in an animal that may be predisposed to the disease but has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), inhibiting the disease (delaying or arresting its development), providing relief from the symptoms or side effects of the disease (including palliative treatment) and relieving the disease (causing the regression of the disease).

[0052] The term "functional deficit" means a behavioral deficit associated with neurological damage. Such deficits include gait deficits as seen in Parkinson's disease patients, motor abnormalities seen in Huntington's disease patients. Functional deficit also includes the abnormal foot placement and memory disorders described in this document.

The terms "G2-MePE" and "NNZ-2566" mean L-glycyl-2-methyl-L-prolyl-L-glutamate.

The term "seizure" means an abnormal pattern of neural activity in the brain that results in a motor deficit or lack of motor control, resulting in abnormal movement, including jerky movement. "Seizure" includes electroencephalogram abnormalities, whether or not accompanied by abnormal motor activity.

[0055] Implicit hydrogen atoms (such as the hydrogen atoms on a pyrrolidine ring, etc.) are omitted from the formulas for clarity, but should be understood to be present.

Autism Spectrum Disorders

[0056] Autism spectrum disorders (ASD) are a set of linked developmental disorders, characterized by abnormalities in social interaction and communication, restricted interests, and repetitive behaviors. The current classification of ASDs recognizes five distinct forms: classic autism or Autism Disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder, and pervasive developmental disorders not otherwise specified (PDD-NOS). The sixth syndrome, pathological demand avoidance (PDA), is yet another pervasive developmental disorder. However, while PDA is increasingly recognized as an ASD, it is not yet part of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), published by the American Psychiatric Association, nor is it is part of the revision proposal, the DSM-V.

Autism

[0057] Classical autism is a highly variable neurological disorder. It is typically diagnosed during infancy or early childhood, with obvious symptoms, often apparent from the age of 6 months, and becoming established by 2-3 years. According to the criteria established in the DSM-IV, the diagnosis of autism requires the presence of a triad of symptoms including (a) impairments in social interaction, (b) impairments in communication and (c) restricted and repetitive interests and behaviors. Other disorders, such as atypical eating, are also common but not essential for diagnosis. Of these deficiencies, social interaction deficiencies are particularly important for diagnosis, and two of the following deficiencies must be present for a diagnosis of autism: (i) deficiencies in the use of multiple nonverbal behaviors (eg. (p, eye contact) to regulate social interaction; (ii) inability to develop developmentally appropriate peer relationships; (iii) lack of spontaneous search to share pleasure, interests or achievements; (iv) lack of social or emotional reciprocity.

[0058] Communication impairments in autism can manifest in one or more of the following ways: delay (or total lack) in the development of spoken language; marked impairment in the ability to initiate or maintain a conversation, stereotyped and repetitive use of language, and/or lack of spontaneous make-believe. Restricted, repetitive, and stereotyped pattern of behavior is also necessary for diagnosis, such as preoccupation with one or more interest considered abnormal in intensity, inflexible adherence to routines or rituals, repetitive motor mannerisms, and/or persistent focus on parts of objects .

[0059] Finally, for the diagnosis of autism, failure in the functioning of at least one area (ie, social interaction, language, or imaginative reproduction) needs to start in less than 3 years of age.

Autism is commonly associated with epilepsy or epileptiform activity on the electroencephalogram (EEG). As many as 60 percent of patients with autism have epileptiform activity on their EEGs (Spence and Schneider, 2009 Ped Res 65: 599-606).

[0061] Autism is also associated with disturbances in the function of IGF-1, which is depleted in the Central Nervous System (CNS) in patients with autism (Rikonen et al., 2006 Devel Med. Child. Neurol 48:751 -755) . IGF-1 levels in the CNS increase in patients with autism after treatment with agents that reduce symptoms, such as fluoxetine (Makkonen et al., 2011 Neu-ropediatrics 42:207-209).

[0062] It is important to emphasize that autism shares characteristics of Rett Syndrome and Fragile X Syndrome in relation to neuronal connectivity. All three disorders are characterized by defects in neuronal function and synaptic connectivity. This is reflected in post-mortem studies of the human brain in this patient group, which all show failure to form normal synaptic connections. This is reflected in altered morphological features, either a reduction in dendritic spine density of neurons, or improved dendritic spine density but associated with immature synapses. This is reflected in animal models of autism, Rett syndrome and fragile X syndrome, which are based on genetic alterations known to be pathological in these disorders. In these animal models, neuronal connectivity defects are revealed morphologically, and also as a Long Term Potentiation (LTP) failure. This is important as IGF-1, IGF-1 [1 -3] and G-2-MePE enhance synapse formation.

Asperger's syndrome

[0063] Asperger's Syndrome or Asperger's Disorder is similar to autism, and shares certain characteristics. Like autism, Asperger's syndrome is also characterized by impaired social interaction and this is accompanied by restricted and repetitive behavior and interests. Thus, the diagnosis of Asperger syndrome is characterized by the same triad of disabilities as autism. However, it differs from other ASDs in that it has no general delay in language or cognitive development and no deficits in interest in the individual's environment. In addition, Asperger's syndrome is generally less severe in symptomatology than classic autism, and Asperger's patients can function self-sufficiently and lead relatively normal lives.

Childhood Disintegrative Disorder

[0064] Childhood Disintegrative Disorder (CDD), also known as Heller's syndrome, is a condition in which children develop normally until the age of 2-4 years (ie, later than autism and Rett syndrome ), but then demonstrate a severe loss of social, communication, and other skills. Childhood Disintegrative Disorder is very similar to autism and both involve normal development followed by a significant loss of language, social play, and motor skills. However, childhood disintegrative disorder usually occurs later than autism, involves a more dramatic loss of skills, and is much less common.

[0065] The diagnosis of CDD is dependent on the dramatic loss of previously acquired skills in two or more of the following areas: language, social skills, play, motor skills (such as a dramatic decline in the ability to walk, climb, grip, etc.), bowel control or bladder control (despite being toilet trained). The loss of developmental skills can be abrupt and take place over days or weeks, or it can be more gradual.

Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS)

[0066] Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS) is an ASD that describes patients who exhibit some, but not all, of the symptoms associated with other well-defined ASDs. The main criteria for diagnosing an ASD include difficulty socializing with others, repetitive behaviors, and heightened sensitivities to certain stimuli. These are all found in the ASDs described above. However, autism, Asperger syndrome, Rett syndrome, and childhood disintegrative disorder all have other features that allow for their specific diagnosis. When the specific diagnosis of one of these four disorders cannot be made but ADS is apparent, a diagnosis of PDD-NOS is made. Such a diagnosis can result from symptoms from an older age that are applicable to other conditions on the spectrum.

Rett Syndrome

[0067] Rett syndrome (RTT) is a neurological disorder that affects almost exclusively women (1 in 10:000 live births). RTT is classified as an autism spectrum disorder (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition - Revised (DSM-IV-R). Approximately 16,000 patients are currently affected by it in the USA {Rett Syndrome Research Trust data ). For a diagnosis of Rett syndrome, the following symptoms are characteristic: impaired development from 6-18 months; deceleration of head growth rate from age between 3 months and 4 years; severely impaired language, repetitive and stereotyped hand movements; and gait abnormalities, eg walking on toes or walking with unsteady and stiff legs. the diagnosis. These include breathing difficulties, EEG abnormalities, seizures, muscle stiffness and spasticity, scoliosis (curvature of the spine), teeth grinding, small hands and feet for height, growth retardation, decreased body fat and muscle mass , abnormal sleep patterns, irritability or restlessness, difficulty chewing and/or swallowing, poor circulation and constipation.

[0068] The onset of RTT is usually between 6-18 months of age, with a deceleration in growth and development rates. This is followed by a regression phase (usually in children aged 1-4 years old), a pseudostationary phase (2-10 years old) and a subsequent progressive progressive motor impairment state. Symptoms of RTT include sudden growth slowdown and regression in language and motor skills including manual movements being replaced by stereotyped movements, autistic features, panic attacks such as sleep cycle disorders, tremors, seizures, respiratory dysfunction (episodic apnea, hyperapnea), apraxia, dystonia, dyskinesia, hypotonia, scoliosis or progressive kyphosis and severe cognitive impairment. Most RTT patients survive to adulthood with severe disabilities and require 24-hour care.

[0069] Between 85% and 95% of TTR cases are reported to be caused by a mutation of the MeCP2 gene (Amir et al., 1999. Nat Genet 23: 185-188; Rett Syndrome Research Trust) - a gene encoding the methyl-CpG- binding protein 2 (MeCP2). Mecp2 maps to the X chromosome (location Xq28) and, for this reason, gene mutations in males are generally lethal. While RTT is a genetic disease, less than 1% of reported cases are hereditary; almost all Mecp2 mutations occur de novo, with two-thirds caused by mutations in eight CpG dinucleotides (R106, R133, T158, R168, R255, R270, R294, and R306), located in the third and fourth exons.

[0070] MeCP2 is a protein that binds methylated CpG dinucleotides to exert transcriptional DNA silencing in the CNS. The main effect of a reduction or absence of MeCP2 appears to be a deficiency in dendritic spine development and synapse formation. MeCP2 expression appears to correlate temporally with brain maturation, explaining why symptoms usually appear at around 18 months of age.

Presentation of Common Characteristics of ASDs

[0071] Taking the ASDs together, it is clear that there are similarities in symptom presentation between all 5 forms. These common features are impairments of normal social skills, and repetitive behaviors. In all but Asperger syndrome, there is also a consistent presentation of retarded intellectual development most commonly manifesting as a deficit in language skills. Cognitive loss in relation to normal parameters for age is often quite pronounced in autism, Rett syndrome, CDD and PDD-NOS. The presence of epilepsy or abnormal EEG activity is also common in autism, Fragile X Syndrome, and Rett Syndrome. Epilepsy arises in situations of abnormal neuronal connectivity. Decreased neuronal connectivity and demented synaptic function is a common feature of autism, Fragile X Syndrome and Rett Syndrome, and animal models of these conditions.

Genetic Models of ASD

[0072] To provide validity, animal models of ASDs must demonstrate symptoms similar to clinical conditions and exhibit a reasonable degree of face validity in relation to the etiology of these symptoms. It is known that classical autism can be caused by many different genetic deficiencies and no single genetic defect is responsible for more than a small percentage of autism cases. Indeed, recent studies have revealed numerous structural de novo variations of chromosomal loci believed to underlie ASD, as well as hereditary genetic abnormalities (Marshall et al., 2008; Sebat et al., 2007). Thus, copy number variation (CNV), translocation and inversion of genetic sequences at 20 key points or more, including 1p, 5q, 7q, 15q, 16p, a 7P and Xq, were mapped as ASD sites.

[0073] However, despite the underlying polygenetics underpinning ADS and the complexity of its etiology, it is known that certain genetic defects can produce ASD. Some of the best characterized defects arise from chromosomal aberration genes that encode a set of postsynaptic density proteins, including neuroliguin-3 (NLGN3), neuroliguin-4 (NLGN4), neuroxin NRXN-la (1) and SHANK3 ( Sebat et al., 2007). Importantly, these defects point to altered synaptic function and therefore disturbed neuronal connectivity as a common final pathway in autism and related disorders (Minshew and Williams 2007 Arch Neurol. 64:945-950; Gilman et al., 2011 Neu-ron.. 70:898-907). Such connectivity deficits are reflected in morphological changes in the post mortem examination, which reveal the increased density of dendritic spines in autism (HUTSLER and Zhang 2010 Brain Res. 1309:83-94).

[0074] NLGN3 and NLGN4 are postsynaptic cell adhesion molecules present in glutamatergic synapses. They play an important role in coordinating presynaptic contact with the postsynaptic site and also interact with the postsynaptic scaffolding protein shank3. Mutations to NLGN3 and NLGN4 have been observed in the ASD population and represent perhaps 1% of all cases of ASD (Lintas & Persico, 2008). Jamain and colleagues first reported a missense mutation for NLGN3 and a frameshift mutation for NLGN4 in two unrelated disciplines, resulting in Asperger syndrome and classical autism respectively (Jamain et al., 2003). Although the incidence of NLGN3 or NLGN4 mutations in the ASD population is low (in fact, not many mutations were observed in a study of 96 autism patients in a Canadian study; Gauthier et al., 2005), it has been confirmed in studies preclinical findings that neuroligin mutations can actually model autistic symptoms. Thus, the introduction of mice from the same missense R451C to NLGN3 that has been clinically reported results in a mutant strain of the mouse showing reduced social interaction and increased inhibitory synaptic transmission (Tabuchi et al., 2007).

[0075] The mouse R451C mutant, therefore, represents a model for ASD based on NLGN3 mutation. In this case, the mutation at position R451 NLGN3 results in the "gain of function" mutation

[0076] In contrast, modeling the clinical mutation of NLGN4 in mice is achieved by a "loss-of-function" mutation of NLGN4 (a classic and knockout model). In this model, mutant mice exhibit a deficit in social interaction and reduced ultrasonic vocalization (Jamain et al., 2008). Communication deficits are critical for clinical ASDs and in mice knocked out with NLGN4 a reduction in the ultrasonic vocalizations of male mice exposed to wild-type female counterparts supports the face validity of the strain as an ASD model.

[0077] Presynaptic neurexin proteins induce postsynaptic differentiation into opposing dendrites through interactions with postsynaptic neurolignin counterparts. Mutations of the neuroxin-lα (NRXNl) gene have been reported in numerous studies (Sebate et al., 2007; Marshall et al., 2008; Kim et al., 2008; Yan et al., 2008) and these have been observed in the form of variants in the number of copies. As with NLGN mutations, when an NRXN gene mutation is introduced into mice (in the form of a knockout gene), a mutant strain with certain characteristics similar to ASD is produced (Etherton et al., 2009). These mice knocked out with NRXNI show a decrease in the frequency of miniature hippocampal excitatory postsynaptic current (mEPSC) and a decreased input-output ratio of evoked currents. These electrophysiological effects are related to decreased excitatory transmission in the hippocampus. In addition to the decrease in excitatory neurotransmission, NRXNI knockout mice show a decrease in prepulse inhibition, although social behavior appears to have been affected (Etherton et al., 2009).

[0078] Sharing certain characteristics with the trans-synaptic construct of neuroxin-NLGN, a cell adhesion molecule I (CADMl) is a protein of the immunoglobulin family presenting both pre- and postsynaptic, which is also involved in the activity of adhesion of synaptic trans-cells (Biederer et al., 2002). CADMI gene mutations have been detected in patients with ASD and appear to represent an additional possible cause of these conditions (Zhiling et al., 2008).

[0079] Analysis of CADMl knocked out mice reveals that these animals show increased anxiety-related behavior, impaired social interaction, and impaired social memory and recognition. In addition, CADMI knockout mice demonstrate the weakest motor skills. (Takayanagi et al., 2010). These dysfunctions are more consistent with the symptomatology of ASD.

The 22ql3 deletion syndrome (also known as Phelan-McDermid Syndrome), is a rare genetic disorder caused by a microdeletion at the q13.3 terminal end of chromosome 22. This microdeletion is rarely discovered by typical genetic screening and a fluorescence in situ hybridization test is recommended to confirm the diagnosis. Recent work indicates that the syndrome is caused by errors in the shank3 gene, a postsynaptic density protein critical for normal neuronal function. Interestingly, errors in this gene have also been associated with ASD and the 22q13 deletion syndrome can commonly lead to a diagnosis of ASD (Durand et al., 2007; Moessner et al., 2007; Sykes et al., 2009). Given the close association of 22q 13 deletion syndrome and the consequent diagnosis of ASD, a mutant mouse model of this mutation was developed.

[0081] The knocked-out shank3 mouse exhibits several deficiencies that mirror the symptoms of ASD, including reduced ultrasonic vocalizations (ie, diminished social communication) as well as decreased social interaction time among mice. In addition, these mice had impaired hippocampal CAI excitatory transmission, as measured by evoked current input-output ratio and impaired long-term potentiation (LTP). It is believed that LTP is a process of formation and consolidation of the underlying physiological memory. Thus, the model exhibits a phenotype similar to the NLGN4 knockout, consistent with ASD.

[0082] As already noted, the 22q 13 deletion syndrome itself is very rare. However, it does provide important information involving specific genes that may be involved in the etiology of ASDs. In addition to shank3, this disorder reveals an additional possible gene defect in ASD. Of the 50 or more cases of 22q 13 deletion syndrome described, all have a deletion of a gene that extends beyond shank3 to include an additional gene known as the islet Brain-2 (IB2) gene (Sebate et al., 2007) . The IB2 protein interacts with many other proteins, including MAP kinases and amyloid precursor protein, appears to influence protein trafficking in neurites, and is enriched with postsynaptic densities (Giza et al., 2010). Mice lacking the protein (IB2-/- knockout mice) exhibit impaired social interaction (reduced social sniffing and interaction time), reduced exploration, and reduced cognitive and motor defects (Giza et al., 2010). This behavioral phenotype has been associated with reduced excitatory transmission in cerebellar cells. As with shank3 knockout, the IB2 mutation phenotype is therefore also consistent with ASD.

[0083] In addition to the animal models of postsynaptic density protein defects described above, other monogenetic syndromes that share several features with ASDs can lead to autism by offering another pathway for drug targeting to ASD. An excellent example of this is Fragile X Syndrome.

Fragile X syndrome (FXS) is caused by the expansion of a single trinucleotide gene sequence (CGG) on the X chromosome that results in the inability to express the protein encoded by the fmrl gene. FMR1 (Mental Retardation X Frail 1) is a protein necessary for normal neural development. FXS can cause a child to have autism (Hagerman et al., 2010), in 2-6% of all children diagnosed with autism the cause is an FXS gene mutation. Furthermore, approximately 30% of SXF children have some degree of autism and more than 30% are diagnosed with PDD-NOS (Hagerman et al., 2010). In fact, Fragile X Syndrome is the most common single-gene known cause of autism. The FMR1 knockout mice were developed as an SXF model and therefore as another ASD model. Knockout mutation of the FMR1 gene has been shown to result in neuronal connectivity deficits, such as abnormal dendritic spine development and pruning (Comery et al., 1997), along with protein dysregulation associated with dendritic scaffold proteins (including stem 1) and glutamate receptor subunits at postsynaptic densities (Schiitt et al., 2009). These effects on dendritic morphology result in deficits in functional measures of connectivity such as decreased LTP in the cortex and amygdala (Zhao et al., 2005) and in the hippocampus (Lauterborn et al., 2007), as well as impaired cognition (Kreuger, et al., 2011) and an increase in social anxiety (Spencer et al., 2005). These connectivity deficits are mirrored in FXS patients, who show greater density of dendritic spines in postmortem analyzes (Irwin et al., 2000 Cereb Cortex 10: 1034-1048). This increased density of dendritic spines is accompanied by immature synapses (Klemmer et al., 2011 J Biol Chem. 286:25495-25504), that is, it may represent a functionally immature state.

[0085] In contrast to autism ASDs, Asperger, CDD and PDD-NOS, Rett syndrome appears to have an almost monogenetic basis and can be modeled in mice with good face validity. It is believed that Rett Syndrome is caused, in up to 96% of cases, by a defect in the MECP2 gene (Zoghbi, 2005). As a result, knockout MeCP2 mutant mice provide an animal model with all the features of clinical Rett syndrome, with a phenotype showing some overlap with NLGN4, shank3 and IB2 knockout models of ASD. Thus, MeCP2 knockout mice exhibit a clear deficiency in LTP in the hippocampus, along with a corresponding decrease in social and spatial memory (Moretti et al., 2006) and impaired object recognition (Schaevitz et al., 2010). This deficiency in LTP is accompanied by a decrease in the density of the dendritic spine. Patients with Rett Syndrome show reduced density of dendritic spines (Beli-chenko et al., 1994 Neuroreport 5: 1509-1513).

[0086] Thus, ASDs in humans, share many features of cognitive or developmental disorders in animals, including rodents. Therefore, studies of ASD therapies in rodents such as mice and rats are reasonably predictive of results obtained in humans. A common feature noted in autism, Fragile X Syndrome, and Rett Syndrome is the presence of neuronal connectivity deficits, reflected in either decreased density of dendritic spines or increased density of dendritic spines with immature synapses. The functional consequences of these morphological changes are similar in animal models of these diseases, which are reflected in LTP deficiencies, for example.

Treatment of ASD and Animal Models with ASD with CIA G-2-MePE

[0087] As described above, a conserved pathology is observed in ASD, which comprises impaired neurite development, impaired synaptic connectivity and a corresponding impairment in social and cognitive functioning as a result. Such synaptic dysfunctions result from genetically altered functions of postsynaptic density proteins. Normal neurite outgrowth and postsynaptic development can be regulated and augmented by growth factors such as brain-derived neurotrophic factor (BDNF; Chapleau et al., 2009) and insulin-like growth factor-1 (IGF-1; Riikonen et al., 2006; Tropea et al., 2009). Indeed, IGF-1 is essential for normal dendritic spine growth and synapse formation (Cheng et al., 2003 J Neurosci Res. 73: -1-9). Medications that promote growth factor function are therefore of use in the treatment of progressive developmental disorders such as ASDs. G-2-MePE is a methylated small molecule analog of the terminal tripeptide of IGF-1, IGF-1 (1 -3). As an IGF-1 mimetic analogue, G-2-MePE exerts trophic and neuroprotective effects in various animal models. G-2-MePE, therefore, is effective in treating the symptoms of ASD, such as those related to synaptic dysfunction resulting from the genetic mutations described above.

[0088] In clinical terms, patients with autism, with autism, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and PDD-NOS, as well as patients with 22q13 deletion syndrome, Fragile X Syndrome and pathological demand avoidance are treated with G-2 MePE. Patients have social and communication impairments, as well as cognitive deficits. It is observed that treatment with G-2-MePE, for example, on a daily basis and in another example, orally, induces an improvement in stereotypic repetitive movements, improves social functioning and improves cognitive performance following if treatment with the drug.

[0089] In animal models of ASDs, daily treatment with G-2-MePE by oral gavage or intraperitoneal injection in the knocked out mice will improve ASD-like symptoms. G-2-MePE is effective in the following ASD mutant mouse models: NLGN3 (R451C) mutant, NLGN4 knocked out, NRXN1 knocked out, CADM1 knocked out, shank3 knocked out, IB2 knocked out, FMR1 knocked out and MeCP2 knocked out. When administered sub-chronically (1 -10 weeks) on a daily basis, G-2-MePE is effective in improving LTP in the hippocampus after rupture stimulation or high frequency stimulation. Likewise, G-2-MePE increases excitatory neurotransmission as measured by electrophysiological recordings of extracellular postsynaptic field potential in the cortex, hippocampus, and cerebellum. As a result of improved excitatory neurotransmission (reversal of the observed ASD-like neurotransmission deficit), G-2-MePE is observed to improve cognitive and motor outcome tests of cognitive performance. Thus, G-2-MePE improves performance in Morris water maze test and arm radial maze test. In social interaction models, G-2-MePE, administered to ASD mutant mice, increases the time spent by knocked out males in social interaction with wild-type females. Furthermore, ultrasonic vocalizations for wild-type female mice are increased. In models where longevity is observed to be reduced in mutant mice compared to wild-type controls (such as the MeCP2 knockout mouse model of Rett syndrome), treatment with G-2-MePE increases the lifespan of the animals .

[0090] G-2-MePE has been shown to inhibit non-convulsive seizures (NCS) in animals with hypoxic-ischemic injury caused by middle cerebral artery occlusion (MCAO; US Patent number 7,714,020, Lu et al., NNZ- 2566, a glypromate analog, attenuates brain ischemia-induced nonconvulsive seisures in rats, 3 Cerebral Blood FLow metabolism (2009) 1-9) and inhibits neuroinflammation in animals with penetrating ballistic injury (pTB1; Wei et al., NNS-2366 treatment Neuroinflammation and pro-inflammatory cytokine expression inhibits induced by experimental penetrating ballistic-like brain injury in rats, J. Neuroinflammation (2009) 6:19, 1-10).

[0091] Our findings that G-2-MePE are also effective in the treatment of Rett syndrome and ASD, which is completely unexpected based on the prior art. This is because the NCS in the MCaO model is caused by hypoxia-ischemia and the expression of inflammatory cytokines in the pTBI model is caused by penetration trauma, which are acute insults, which are very different from the chronic effects of MECP2 or other mutations in synaptic maturation.

[0092] Since G-2-MePE is an element of the GPE analog compounds disclosed herein, any of the compounds described may also be effective in treating the symptoms of ASDs. Furthermore, since the compounds and methods of this invention address underlying neurological mechanisms (eg, decreasing neural inflammation through inhibition of inflammatory cytokine release), the present invention may provide more than short-term symptom monitoring. In contrast, compounds and methods of the present invention can improve neuronal function, promote neuronal cell migration, promote neurogenesis, promote neuronal stem cell differentiation, promote axonal and dendritic growth, and promote synaptic transmission , thus alleviating the adverse symptoms of ASDs.

Compounds of the Invention

[0093] Although the broadest definition of the invention is defined in the Summary, certain compounds of the present invention are described herein.

em que m é 0 ou 1; n é 0 ou 1; X é H ou -NR6 R7; Y é H, alquila, - CO2R5, ou -CONR6 R7; Z é H, alquila, - CO2R5 ou -CONR6 R7; R1 é H, alquila ou aralquila; R2, R3, e R4 são, independentemente, H ou alquila; cada R5 é independentemente H, alquila, ou um resíduo de álcool graxo ; cada R6 e R7 é independentemente H, alquila, ou aralquila, ou -NR6 R7 é pir- rolidina, piperidina, ou morfolino; ou uma lactona formada quando um composto em que Y é -CO2 (alquila), e Z é -CO2H ou onde Y é - CO2H e Z é -CO2 (alquila) é lactonizado; e os seus sais farmaceuticamente aceitáveis, desde que o composto não seja GPE, N-Me-GPE, amida de GPE, APE, GPQ ou um seu sal.[0094] In one aspect, this invention provides compounds of formula 1 and formula 2:

where m is 0 or 1; n is 0 or 1; X is H or -NR6 R7; Y is H, alkyl, -CO2R5, or -CONR6 R7; Z is H, alkyl, -CO2R5 or -CONR6 R7; R1 is H, alkyl or aralkyl; R2, R3, and R4 are independently H or alkyl; each R5 is independently H, alkyl, or a fatty alcohol residue; each R6 and R7 is independently H, alkyl, or aralkyl, or -NR6 R7 is pyrrolidine, piperidine, or morpholino; or a lactone formed when a compound where Y is -CO2 (alkyl), and Z is -CO2H or where Y is -CO2H and Z is -CO2 (alkyl) is lactonized; and pharmaceutically acceptable salts thereof, provided that the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt thereof.

In some aspects, the present invention includes: (a) the compounds are compounds of formula 1; (b) m is 0; (c) n is 1; (d) at least one of X, Y, R1, R2, R3, R4 and R5 is not hydrogen; (e) X is -NR6 R7; and (f) Y is -CO2R5 or -CO2NR6 R7; and (g) Z is -CO2R5 or -CO2NR6 R7.

[0096] Other compounds of the invention are compounds of formula 1 wherein X is -NR6 R7 and R6 and R7 are independently alkyl or aralkyl. The most preferred embodiment is a compound of formula I wherein X is -NR6 R7 and both R6 and R7 are alkyl.

[0097] Yet another compound of the invention is G-2-MePE, a compound of formula 1, wherein m is 0, n is 1, R1 = R3 = R4 = H. R2 is methyl, X is NR6 R7 where R6 = R7 = H, Y is CO2R5, where R5 = H, Z is CO2R5, where R5 = H.

Pharmacology and Utility

The compounds of this invention may have anti-inflammatory, anti-apoptotic, anti-necrotic and neuroprotective effects. Its in vivo activity can be measured by cell counting, specific staining of the desired markers, or by methods such as those discussed in Klempt ND et al.: hypoxia-ischemia induces transforming growth factor β1 mRNA in infant mouse brain . Molecular Brain Research: 13:93-101. Its activity can also be measured in vitro using methods known in the art or described herein.

[0099] Conditions that affect brain function become prevalent in an aging population. Memory loss and memory loss are distressing for affected patients and their families. Memory loss or impairment can result from normal aging, brain damage, neurodegenerative diseases, and psychiatric or psychological disorders. Therefore, it is of great benefit to patients, their families and society that new compounds are identified and characterized which improve memory and/or cognitive function and treat or prevent memory loss or impairment.

[00100] It is desirable to study the potential effects of therapeutic agents in animal systems. Such a useful system is the mouse. It is known that, with aging, mice and other animals (including humans) can show symptoms of memory loss, memory dysfunction and other cognitive dysfunctions. Furthermore, rat studies of therapeutic agents are known to be predictive of therapeutic effects in humans. Thus, studies of the effects of GPE and G-2-MePE and cognitive function in elderly rats are reasonably predictive of the therapeutic effects of these agents in the aging of human beings who have or are prone to acquire memory deficits or other dysfunctions. cognitive. The compounds of the present invention can improve cognitive function and/or treat memory disorders. Cognitive enhancement activity and therapeutic activity in vivo can be measured by standard neuropsychological or behavioral tests known to those skilled in the art. Such tests can be chosen from the wide range of available tests described above, and will vary depending on the cognitive function being tested and the condition of the animal.

[00101] Standard behavioral tests useful for testing cognitive function in animal models include, but are not limited to the Morris water maze test, passive avoidance response test, new object recognition test, olfactory discrimination test, 8-arm radial maze test and the T-maze test. These tests are directly applicable to studies of the effects of GPE and G-2-MePE on cognitive function in elderly rats.

[00102] The compounds of this invention are also expected to have pharmacological and therapeutic activities similar to those of GPE, and these activities can be measured by methods known in the art, and discussed in the documents cited herein, and the methods used to measure the activity of the GPE.

[00103] The therapeutic ratio of a compound can be determined, for example, by comparing the dose that provides effective anti-inflammatory, anti-apoptotic and anti-necrosis activity in a suitable in vivo model such as a hypoxic-ischemic lesion (Sirimanne ES , Guan J. Williams CE and Gluckman PD: Two models for determining injury and repair mechanisms after hypoxic-ischemic injury in the developing rat brain (Journal of Neuroscience Me-thods: 55:7-14, 1994), in a suitable animal species, such as the rat, at the dose that provides significant side effects in the test animal species.

[00104] The therapeutic ratio of a compound can also be determined, for example, by comparing the dose that provides effective improvement in cognitive function or treats a memory disorder in a suitable in vivo model (Examples 4, 5 and 6 below), in a suitable animal species, such as a rat, at the dose that provides the significant weight loss (or other observable side effects) in the test animal species.

The compounds of this invention may be useful in the treatment of a variety of neurodegenerative disorders, including hypoxia/ischemia and neuronal degeneration (US Patent number 7,041,314), traumatic brain injury, motor disorders and seizures, stroke , and cardiac revascularization surgery (US Patent number 7,605,177), non-convulsive attacks (US Patent number 7,714,020) and cognitive function disorders (US Patent number 12/903,844). Furthermore, as described more fully hereinafter, the compounds of the present invention may be useful for the treatment of Rett syndrome, including prolonging life, increasing neuronal activity, and treating seizures associated with Rett syndrome.

[00106] In a study of Rett Syndrome in mice (using the knockout MeCP2 model), it was found that GPE was has life-prolonging effect and increased neuronal function (US Publication number 2009/0099077). However, as further described herein, GPE, being a naturally occurring peptide, is rapidly degraded in vivo and in vitro, and its usefulness in the chronic therapy of patients with Rett syndrome is therefore uncertain. .

Pharmaceutical Compositions and Administration

[00107] In general, the compounds of this invention may be administered in therapeutically effective amounts, in any of the usual ways known in the art, either alone or in combination with at least one other compound of this invention and/or at least one other therapeutic agent conventional for the disease to be treated. A therapeutically effective amount can vary widely depending on the disease or injury, the severity of the disease, the age and relative health of the animal being treated, the potency of the compound(s), and other factors. As a result, therapeutically effective anti-inflammatory, anti-apoptotic, anti-necrotic, anti-neurodegenerative amounts of compounds of the present invention can range from about 0.001 milligrams per kilogram (mg/kg) to about 100 (mg/kg) of the animal, for example, about 0.1 to about 10 mg/kg, with lower doses, such as about 0.001 to about 0.1 mg/kg, for example about 0.01 mg/kg, being suitable for administration via of the cerebrospinal fluid, such as by intracerebroventricular administration, and higher doses, such as about 1 to about 100 mg kg, for example about 10 mg kg, being suitable for administration by methods, such as orally , systemic (eg transdermal), or parenteral (eg intravenous). A person of ordinary skill in the art will be able, without undue experimentation, having regard to their ability and this disclosure, to determine a therapeutically effective amount of a compound of the present invention for a particular disease or injury.

[00108] In general, the compounds of this invention may be administered as pharmaceutical compositions by one of the following routes: oral, topical, systemic (eg, transdermal, intranasal or by suppository), or parenteral (eg, intramuscularly, subcutaneously or intravenous) by administration to the central nervous system (eg, by intraspinal or intercisternal injection); by implantation, and by infusion through devices such as osmotic pumps, implantable pumps, transdermal patches, and the like. Compositions may take the form of tablets, pills, capsules, semi-solids, powders, extended release formulations, solutions, suspensions, elixirs, aerosols, soluble gels or any other suitable compositions and comprise at least one compound of the present invention in combination with at least one minus one pharmaceutically or physiologically acceptable excipient.

[00109] Suitable excipients are well known to those skilled in the common art and they, and the methods of formulating the compositions, can be found in standard references such as, Gennaro AR: Remington: The Science and Practice of Pharmacy, 20"'ed ., Lippincott, Williams & Wilkins, 2000. Suitable liquid vehicles, especially for injectable solutions, include water, aqueous brine solution, aqueous dextrose solution, glycols, and the like, with isotonic solutions being preferred for intravenous, intraspinal administration. , intracystic and administration and vehicles, such as artificial cerebrospinal fluid, are also especially suitable for the administration of the compound to the CNS. The above text is expressly incorporated in its entirety by reference.

The compounds of the present invention can be administered orally, in tablets or capsules. In some embodiments, the compounds of this invention can be prepared in water-in-oil emulsions, in the form of microemulsions, coarse emulsions, liquid crystals, or nanocapsules (US Application Number 12/283 of 684, now US Application Number 7,887,839, granted in February 15, 1201). Since the compounds of the present invention can exhibit substantially oral bioavailability, they can be advantageously used for convenient chronic administration. In addition, available oral compositions include soluble hydrogels that contain active compounds, thus allowing oral administration of neuroprotective compounds without the need for a patient to swallow a tablet or capsule. These slow release materials and gels are known in the art.

The compounds of this invention may be administered before or after the onset of a condition that is likely to cause neurodegeneration or a symptom thereof. For example, it is known that hypoxia/ischemia can occur during coronary artery bypass graft (CABG) surgery. Thus, a patient may be pretreated with a compound of the present invention prior to being placed in an extracorporeal oxygenation system. In some embodiments, it may be desirable to administer a compound of the present invention beginning about 4 hours before surgery, or before an event, which is likely to lead to a traumatic neurological injury or other injury. In other embodiments, it may be desirable to infuse a compound of the present invention during surgery or during the surgical procedure to repair neurological damage. The compounds of the present invention can also be used in emergency situations, for example, in a patient who has only experienced a stroke, hypoxic event, traumatic brain injury or other acute insult. In such situations, a compound of the present invention can be administered immediately after a diagnosis of nerve damage is made.

[00112] In some situations, kits containing the compound of the present invention can be prepared before use in the field. A kit may contain a vial containing a compound of the invention in a pharmaceutically acceptable formulation (eg, for injection or oral administration), together with a syringe or other delivery device, and instructions for use. In situations where a seizure is diagnosed, a compound of the present invention may be administered along with an anticonvulsant. Many anticonvulsants are known in the art and need not be described in detail herein.

[00113] In addition, "secondary" neurological injuries can occur following a primary insult, such as a traumatic injury, stroke, or surgical procedure. For example, after a stroke, penetrating brain injury, or a CABG procedure, neural tissue inflammation can lead to neurodegeneration. Secondary damage may be reflected by increased activation of inflammatory cells (eg, astrocytes and/or microglia), and actions of inflammatory mediators may cause neurological damage. Thus, in some embodiments, it may be desirable to administer a compound of the present invention in periods prior to the insult and up to about 100 hours after the insult. In other embodiments, it may be desirable to administer a compound of the present invention beginning before the insult, during the insult and after the insult, either continuously, as an infusion, or in doses separated by a desired time interval.

[00114] The compounds of this invention can also be suitably administered by a controlled release system or gel material with G-2-MePE incorporated therein. Suitable examples of sustained release compositions include semi-permeable polymer matrices in the form of molded articles, for example, films or microcapsules. Extended-release matrices include polylatides (US application number 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983), poly(2-methacrylate). -hydroxyethyl) (Langer et al., 1981), ethylene vinyl acetate (Langer et al., above), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). In addition, gel compositions based on polysaccharides (eg carboxymethyl cellulose, carboxyethyl cellulose, chitosan or other cellulose derivatives) and polyethylene oxide derivatives (eg polyethylene glycols) can be used. These gel compositions are soluble in aqueous solutions, are biocompatible, non-toxic, and therefore can be used to deliver compounds of the present invention to any mucosal surface, including the oral cavity, oropharynx, urogenital tract, intestine, or rectum.

[00115] Sustained-release compositions also include a compound entrapped in liposomes. Liposomes containing the compound are prepared by methods known per se: DE 3,218,121; Epstein et al., 1985; Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949, EP 142,641, Japanese Patent Application 83-118008; Patent Applications numbers 4,485,045 and 4,544,545 and EP 102,324. Typically, liposomes are of the small (from at or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted for therapy more efficient. Each of the above-identified publications is hereby expressly incorporated by reference in their entirety, as if each had been so incorporated separately.

The compounds of the present invention can also be linked to polyethylene glycol ("PEGylated") to increase its lifetime in vivo, based, for example, on the conjugate technology described in WO 95/32003.

Desirably, if possible, when administered as an anti-inflammatory, an anti-apoptotic agent, an anti-necrotic agent, or an anti-neurodegenerative agent, the compounds of the present invention may be administered orally. The amount of a compound of the present invention in the composition can vary widely depending on the type of composition, size of a dosage unit, species of excipient and other factors well known to those skilled in the art. In general, the final composition may comprise about 0.0001 percent by weight (% by weight) to about 10% by weight of the compound of the present invention, preferably about 0.001% by weight to about 1% by weight. , with the remainder being an excipient or excipients.

[00118] The composition may optionally contain, in addition to a compound of the present invention, at least one agent selected from, for example, growth factors and their associated derivatives (insulin-like growth factor I (IGF- I), insulin-like growth factor II (1GF-II), transforming growth factor-β1, activin, growth hormone, nerve growth factor, brain-derived neurotrophic factor (BDNF), growth hormone, protein binding to IGF binding proteins (IGFBP-3 especially), basic fibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF -6, keratinocyte growth factor, androgen-induced growth factor. Other elements of the FGF family include, for example, int-2, homologous to fibroblast growth factor 1 (FHF-1), FHF-2, FHF- 3 and FHF-4, keratinocyte growth factor 2, cell activating factor glial, FGF-10 and FGF-16, ciliary neurotrophic factor, brain-derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial cell lineage-derived neurotrophic factor, factor activity-dependent neurotrophic, leukemia inhibitory factor, cytokine, oncotatin M, interleukin), α, β, Y or consensus inferon and TNF-α. Other forms of neuroprotective therapeutic agents include, for example, clomethiazole, kynurenic acid, Semax, tacrolimus, L-threo-P-phenyl-2-decanoylamino-3-morpholine-1-propanol, andrenocorticotropin(4-9) analog [ORG 2766] and dizolcipin (MK-801), selegiline, glutamate antagonists such as mematin (Namenda) NPS1506, GV 105260, MK-801, GV150526; AMPA antagonists such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164, anti-inflammatory agents directed against the addressin MAdCAM-1 and/or its de-integrates α4 receptors (α4β1 and α4β7), such as the anti-MAdCAM-1 mAb MECA-367 (ATCC accession number HB-9478). Combination therapy with metabotropic glutamate receptor antagonists such as fenobam may also be useful. Furthermore, in addition to a compound of the present invention, a composition may include a selective serotonin reuptake inhibitor such as fluoxetine, a selective serotonin reuptake norepinephrine such as viloxazine, or an atypical antipsychotic such as risperidone. Most of these agents, particularly peptides such as growth factors, etc., are not orally active, and will need administration by injection or infusion.

Preparation of Compositions

The starting materials and reagents used in preparing these compounds are either available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, Wisconsin), Bachem (Torrance, California), Sigma (St. Louis, MO), or are prepared by methods well known to one of ordinary skill in the general art following the procedures described in the references, such as, Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y. 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989; Organic Reactions, volumes 1-40, John Wiley and Sons, New York, N.Y. 1991; March J.; Advanced Organic Chemistry, 4th ed. John Wiley and Sons, New York, NY, 1992, and Larock: Comprehensive Organic Transformations, VCH Publishers, 1989. In most cases, amino acids and their esters or amides and protected amino acids are widely available commercially , and the preparation of modified amino acids and their esters or amides is widely described in the chemical and biochemical literature and thus well known to those skilled in the common art. For example, N-pyrrolidine acetic acid is described in Dega-Szafran Z and Pryzbylak R. Synthesis, IR, and from NMR studies of α-(1-pyrrolidine) zwitterionic alkanecarboxylic acids and their N-methyl derivatives. J. Mol. Struct:. 436-7, 107-121, 1997, and N-piperidineacetic acid is described in Matsuda S, Ito S and Sekiya M. The reaction of N-(alkoxymethyl)dialkylamines with isocyanides. Chem. Pharm. Bull.: 23 (1), 219-221, 1975. Each of the above-identified publications are expressly incorporated herein by reference as if they were individually incorporated.

[00120] The starting materials, intermediates and compounds of this invention can be isolated and purified using conventional techniques, including filtration, distillation, crystallization, chromatography and the like. They can be characterized using conventional methods, including physical constants and spectral data.

The compounds of this invention can be prepared by the methods described below and as determined in the Examples.

The compounds of formula 1 are the analogues of GPE, or modifications thereof, such as esters or amides. In general, they can be prepared by methods such as those which are already well known to those skilled in the common art of peptide and modified peptide synthesis, following the reaction schemes set out in Figures 1-11 accompanying this described report. active or following other methods well known to those skilled in the art of synthesizing peptides and analogues.

[00123] Conveniently, the synthetic production of polypeptides of the invention can be according to the solid phase synthesis method described by Merrifield. and others. The synthesis of a tetrapeptide: J. Amer. Chem. Soc: 85, 214921 56, 1963. This technique is well understood and is a common method for preparing peptides. The general concept of this method depends on linking the first amino acid in the chain to a solid polymer by a covalent bond. Protected amino acids in succession are added, one at a time (step strategy), or in blocks (segment strategy), until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved. By this procedure, the reagents and by-products are removed by filtration, thus eliminating the need for purification of the intermediates.

[00124] Amino acids can be attached to any suitable polymer such as a resin. The resin must contain a functional group to which the first protected amino acid can be securely attached via a covalent bond. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethyl methacrylate and polystyrene. Suitable resins are commercially available and are well known to those skilled in the art. Suitable protecting groups usable in such a synthesis include tert-butyloxycarbonyl (BOC), benzyl (Bzl), t-amyloxycarbonyl (AOC), tosyl (Tos), o-bromo-phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BzlCl2) and phenylmethoxycarbonyl (Z or CBZ). Additional protecting groups are identified in Merrifield, cited above, as well as in Me-Omie JFW: Protective Groups in Organic Chemistry, Plenum Press, New York, 1973, both references expressly incorporated herein in their entirety.

[00125] General procedures for the preparation of peptides of the present invention involve, initially, attachment of a protected amino acid at the carboxy terminus to the resin. After the resin attachment is filtered, washed and the protecting group (desirably BOC) on the I-amino group of the carboxyl-terminal amino acid is removed. Removal of this protective group must, of course, be carried out without breaking the bond between the amino acid and the resin. The next amino, and, if necessary, side-chain protected amino acid, is then coupled to the free I-amino group of the amino acid on the resin. This coupling occurs through the formation of an amide bond between the free carboxyl group of the second amino acid and the amino group of the first amino acid bound to the resin. This sequence of events is repeated with successive amino acids until all amino acids are bound to the resin. Finally, the protected peptide is cleaved from the resin and the protecting groups removed to reveal the desired peptide. The cleavage techniques used to separate the peptide from the resin and to remove the protecting groups depend on the selection of the resin and the protecting groups and are known to those skilled in the art of peptide synthesis.

[00126] Alternative techniques for peptide synthesis are described in Bodanszky et al., Peptide Synthesis, 2nd ed, John Wiley and Sons, New York, 1976. For example, the peptides of the invention can also be synthesized using synthetic methodologies of peptides in standard solution, involving either the stepwise coupling of either amino acid blocks or peptide fragments through enzymatic or chemical amide bond formation methods. (See, for example, HD Jakubke in the Peptides, Analysis Synthesis, Biology, Aca-demic Press, New York, 1987, p.103-165; JD Glass, ibid, pp. 167-184, . and European Patent 0324659 A2 , which describes enzymatic peptide synthesis methods). Such solution synthesis methods are well known in the art. Each of the above-identified publications is expressly incorporated herein in its entirety by reference, as if individually incorporated herein.

[00127] Commercial peptide synthesizers, such as the Applied Biosystems, Model 430A, are available to practice these methods.

[00128] One of ordinary skill in the art will not have to make undue experimentation having regard to the skill and knowledge available in that disclosure, in developing one or more of the synthetic methods suitable for the compounds of the present invention.

[00129] For example, analogs in which the glycine residue of GPE is replaced by an alternative amino acid, or by a non-amino acid, can be conveniently prepared by the methods of preparing a C-protected glutamic acid-proline dipeptide (such as the dibenzyl ester) and coupling that dipeptide with an N-protected glycine analog, such as, BOC-N-methylglycine, BOC-L-valine, N-pyrrolidine acetic acid, and the like, followed by deprotection, such as illustrated in Figures 2 and 3. Analogs in which the glutamic acid residue of GPE is replaced by an alternative amino acid or an amino acid amide or ester can be conveniently prepared by preparing an N-protected glycine-L-proline dipeptide (such as as BOC-glycyl-L-proline) and coupling that dipeptide with a C-protected glutamic acid or analogue thereof such as tert-butyl Y-aminobutyrate, methyl 4-amino-4-dimethylcarbamoyl butyrate, L-glutamine ester methyl, 1-methiglutamate, etc. The lactones can be prepared by the methods of preparing an appropriate mono-ester mono-acid derivative and reducing analogs, in which R2 is alkyl, can be conveniently prepared simply by using the appropriate 2-alkylproline in the synthesis and similar analogs in which R3 represents an alkyl group can be conveniently prepared using N-alkylglutamic acid or the like in the synthesis. When modifications must be made to two or more amino acids, the coupling techniques will still be the same, with only more than one modified amino acid or analog being employed in the synthesis. The choice of appropriate protecting groups for the chosen method (solid-phase or solution-phase), and appropriate substrates, if solid-phase synthesis is used will be within the capabilities of one of ordinary skill in the art.

[00130] The compounds of formula 2 can be prepared from suitably protected 5-oxo-L-proline or its analogues or derivatives, according to methods such as coupling the carboxyl group of proline with a protected glutamic acid or analogue or derivative, to give an analogue of the intermediate A of figure 2, comparable to the coupling reaction shown in figure 2, and then alkylating the pyrrolidine nitrogen with a group of formula A- ---(CH2) m -CH(R1)---CH2R, protected if necessary, where R is a leaving group under alkylation conditions. Alternatively, the suitably protected 5-oxo-L-proline can be first alkylated at the pyrrolidine nitrogen to produce an analogue of intermediate B of Figure 4, and then coupling this with a suitably protected glutamic acid or analogue. or derived as shown in figures 4 to 9.

EXAMPLES

[00131] The following examples are intended to illustrate embodiments of the present invention, and are not intended to limit the scope to those specific examples. Those of ordinary skill in the art can apply the disclosures and teachings disclosed herein to develop other embodiments, without undue experimentation and with a likelihood of success, all such embodiments being considered as part of this invention.

Example 1: Synthesis of N,N-dimethylglycyl-L-prolyl)-L-glutamic acid

[00132] The following non-limiting example illustrates the synthesis of a compound of the invention, N,N-dimethylglycyl-L-prolyl-L-glutamic acid

[00133] All starting materials and other reagents were purchased from Aldrich; BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-L-proline-(e-benzyl)-L-glutamic acid benzyl ester

[00134] To a BOC-proline solution [Anderson GW and McGregor AC: J. Amer. Chem. Soc: 79, 6810, 1994] (10 mmol) in dichloromethane (50 mL), cooled to 0 °C, triethylamine (1.39 mL, 10 mmol) and ethyl chloroformate (0.96 mL, 10 mmol) were added . The resulting mixture was stirred at 0°C for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol) was then added and the mixture stirred at 0°C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate solution and citric acid (2 mol I1), then dried (MgSO4) and concentrated under reduced pressure to give BOC-L-proline-L-glutamic acid dibenzyl ester, ( 5.0 g, 95%).

L-Proline-L-Glutamic Acid Dibenzyl Ester

[00135] A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol), cooled to 0°C, was treated with trifluoroacetic acid (25 mL) for 2 hours at temperature environment. After removing volatiles under reduced pressure the residue was triturated with ether to give L-proline-L-glutamic acid dibenzyl ester.

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

[00136] A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 mL) was added to a stirred and cooled (0°C) solution of L-proline-L-glutamic acid dibenzyl ester (10 mmol) , N,N-dimethylglycine (10mmol) and triethylamine (10.3mmol) in dichloromethane (30ml). The mixture was stirred at 0°C overnight and then at room temperature for 3 hours. After filtration, the filtrate was evaporated under reduced pressure. The resulting crude dibenzyl ester product was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on carbon (0.5 g) and then hydrogenated at room temperature and pressure, until hydrogen absorption has ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to obtain the tripeptide derivative.

[00137] It can be noted that by following the method of the Examples and using alternative amino acids or their amides or esters other compounds of Formula 1 will be produced. Example 2: Synthesis of Glycyl-L-2-methylI-L-prolyl- L-Glutamate Glycyl-L-2-Methylprolyl-L-Glutamic Acid (G-2M and PE)

[00138] Scheme 1, reagents, conditions and yields, (i) SOCl2, MeOH, 79°C, N2, 24 hours (104%), (ii) Et3N, DCC, CH2Cl2, 0°C to room temperature, N2 , 20 hours, (iii) 1M aqueous NaOH, 1,4-dioxane, 19 hours (60%, 2 steps), (iv) Et3N, BoPCI, CH2Cl2, Room temperature, N2, 17 hours (89%), (v ) H2, 10% Pd/C, 91:9 MeOH-H2O, Room temperature, 23 hours (86%).

[00139] L-2-ethylproline and L-glutamic acid dibenzyl ester were purchased from Bachem, N-benzylaxycarbonyl-glycine from Acros Organics and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCI, 97% ) from Aldrich Chem.Co.

Methyl L-2-methylprolinate hydrochloride 2

[00140] Thionyl chloride (5.84 cm3, 80.1 mmol) was carefully added dropwise to a stirred solution of (L)-2-methylproline 1 (0.43 g, 3.33 mmol) ) in anhydrous methanol (30 cm 3 ) at -5°C under a nitrogen atmosphere. The reaction mixture was heated under reflux for 24 hours and the resulting pale yellow solution was concentrated to dryness in vacuo. The residue was dissolved in a 1:1 mixture of methanol and toluene (30 cm 3 ) then concentrated to dryness to remove residual thionyl chloride. This procedure was repeated two more times, yielding hydrochloride 2 (0.62 g, 104%) as a hygroscopic off-white solid, spectroscopically pure: melting point: 127-131°C; [α]D -59.8 (c 0.24 in CH2Cl2); vmax (film)/cm-1 3579, 3398 br, 2885, 2717, 2681, 2623, 2507, 1743, 1584, 1447, 1432, 1374, 1317, 1294, 1237, 1212, 1172, 1123, 981 , 894, 861 and 764; δH (300 MHz; CDCl3; Me4Si) 1.88 (3H, s, Proα-CH3), 1.70-2.30 (3H, br m, Proβ-HAHB, 2.30-2.60 (1H, br m, Proβ-HAHB), 3.40-3.84 (2H, br m, Proβ-H2), 3.87 (3H, s, CO2CH3), 9.43 (1H, br s, NH) and 10.49 (1H, br s, HCl); (75 MHz; CDCl3) 21.1 (CH3, Proα-CH3), 22.4 (CH2, Proy-C), 35.6 (CH2, Proβ-C) .45.2 (CH2, Proδ-C), 53.7 (CH3, CO2CH3), 68.4 (quat., Proα-C) and 170.7 (quat., CO); m/z (FAB+) 323 .1745 [M2.H35Cl.H+: (C7H13NO2)2. H35CI.H requires 323.1738] and 325.1718 [M2.H35CLH+: (C7H13NO2)2. H37CI.H requires 325, 708].

N-benzyloxycarbonyl-glycyl-L-2-methylproline 5

Anhydrous triethylamine (0.45 cm3, 3.23 mmol) was added dropwise to a mixture of methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34 mmol) and N -Benzyloxycarbonyl-glycine (98.5%) 3 (0.52 g, 2.45 mmol) in methylene chloride (16 cm 3 ) at 0°C under an atmosphere of nitrogen. The resulting solution was stirred for 20 minutes and to a solution of 1,3-dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride (8 cm 3 ) at 0°C was added dropwise and the reaction mixture was warmed to room temperature and stirred for a further 20 hours. The resulting white mixture was filtered through a pad of Celite™ to partially remove 1,3-dicyclohexylurea, and the pad was washed with methylene chloride (50 cm3). The filtrate was washed successively with 10% aqueous hydrochloric acid (50 cm 3 ) and saturated aqueous sodium hydrogen carbonate solution (50 cm 3 ), dried (MgSO 4 ), filtered and concentrated to dryness in vacuo. Purification of the residue by flash column chromatography (35 g SiO 2 , 30-70% ethyl acetate - hexane; gradient elution) tentatively gave methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 4 ( 0.56 g), containing 1,3-dicyclohexylurea, as a white semi-solid: Rf 0.65 (EtOAc): m/z (E1+) 334.1534 (M+, C17H22N2O5 requires 334.1529) and 224 (1 ,3-dicyclohexylurea).

To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33 cm3) was added aqueous sodium hydroxide (10 cm3, 10 mmol) dropwise ) and the mixture was stirred for 19 hours at room temperature. Methylene chloride (100 cm3) was then added and the organic layer extracted with saturated sodium hydrogen carbonate (2 x 100 cm3). The combined aqueous layers were carefully acidified with hydrochloric acid (32%), extracted with methylene chloride (2 x 100 cm 3 ), and the combined organic layers were dried (MgSO 4 ), filtered and concentrated to dryness in vacuo. Purification of the resulting residue (0.47 g) by flash column chromatography (17 g SiO 2 , 50% ethyl acetate - hexane to methanol - 30% dichloromethane, gradient elution) provided N-protected dipeptide 5 (0, 45 g, 60%) as a white foam in two steps from hydrochloride 2. Dipeptide 5 was shown to be exclusively trans oriented conformer by NMR analysis: Rf 0.50 (20% MeOH - CH2Cl2), [α ]D -62.3 (c 0.20 in CH2Cl2), vmax (film)/cm-1 3583, 332 4br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251, 1219, 1179, 1053, 1027, 965, 912, 735 and 698; δH (300 MHz; CDCl3, Me4Si) 1.59 (3H, s, Proα-CH3), 1.89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Proβ-HAHB), 2 .01 (2H, dtt, J 18.7, 6.2 and 6.2, Proy-H2), 2.25-2.40 (1H, m, proβ-HAHB), 3.54 (2H, t, J 6.6, Pr β-H2), 3.89 (1H, dd, J 17.1 and 3.9, glyα-HAHB), 4.04 (1H, dd, J 17.2 and 5.3, glyα-HAHB), 5.11 (2H, s, OCH2Ph), 5.84 (1H, br, t, J4.2, NH), 7.22-7.43 (5H m, Ph) and 7.89 (1H, br, -COOH), δC (75 MHz, CDCl3) 21.3 (CH3, Proα-CH3), 23.8 (CH2) ProY-C), 38.2 (CH2, Proβ-C), 43 .6 (CH2, Glyα-C), 47.2 (CH2, Pro'-C), 66.7 (quat, Proα-C), 66.8 (CH2, OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4 (CH, Ph), 136.4 (quat., Ph), 156.4 (quat., NCO2), 167.5 (quat., Gly-CON) and 176.7 (quat., CO): m/z (EI+) 320.1368 (M+.. C16H20N2O5 requires 320.1372).

Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate 7

[00143] Triethylamine (0.50 cm3, 3.59 mmol) was added dropwise to a solution of dipeptide 5 (0.36 g, 1.12 mmol) and p-L-glutamic acid dibenzyl ester of p- toluenesulfonate 6 (0.73 g, 1 0.46 mmol) in methylene chloride (60 cm 3 ) under nitrogen at room temperature and the reaction mixture stirred for 10 minutes. Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) (0.37 g, 1.41 mmol) was added and the colorless solution was stirred for 17 hours. The methylene chloride solution was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate solution (50 cm3), dried (MgSO4), filtered and evaporated to dryness in vacuo. Purification of the resulting residue with repeated flash column chromatography (2 times) (24 g SiO 2 , 3070% ethyl acetate - hexane; elution gradient) afforded a fully protected tripeptide 7 (0.63 g, 89% ) as a colorless oil. Tripeptide 7 was shown to be exclusively the trans oriented conformer by NMR analysis: Rf 0.55 (EtOAc); [α]D -41.9 (c 0.29 in CH2Cl2), vmax (film)/cm-1 583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and 697; δH (400 MHz, CDCl3, Me4Si) 1.64 (3H, s, Proα-CH3), 1.72 (1H, dt, J 12.8, 7.6 and 7.6, Proβ-HAHB) 1, 92 (2H, 5 lines, J 6.7, ProY—H2), 2.04 (1H, 6 lines, J 7.3 Gluβ-HAHB), 2.17-2.27 (H, m, Gluβ-HAHB ), 2.35 -2.51 (3H, m, Proβ-HAHB and GIUY-H2), 3.37-3.57 (2H, m, Proδ-H2), 3.90 (1H, dd, J 17 .0 and 3.6, GlyαHAHB), 4.00 (1H, dd, J 17.1 and 5.1, GliyαHAHB), 4.56 (1H, J 7.7 and 4.9, Gluα-H), 5.05-5.20 (6H, m, 3 x OCH2Ph), 5.66-5.72 (1H,br m, Gly-NH), 7.26-7.37 (15H, m, 3 x Ph ) and 7.44 (1H, d, J 7.2, Glu-NH); δC (100 MHz, CDCl3) 21.9 (CH3, Proα-CH3), 23.4 (CH2,. Proy-C), 26.6 (CH2, Gluβ-C), 30.1 (CH2, Gluy-C ), 38.3 (CH2, Proβ-C), 43.9 (CH2, Glyα-C), 47.6 (CH2, Proδ-C), 52.2 (CH, Gluα-C), 66.4 ( CH2, OCH2Ph), 66.8 (CH2, OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat, Proα-C), 127.9 (CH, Ph), 128.0 (CH, Ph ), 128.1 (CH, Ph), 128.2 (CH, Ph), 128.2 (CH, Ph), 128.3 (CH, Ph), 128.4 (CH, Ph), 128.5 (CH, Ph), 128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.1 (quat., Ph). ., NCO2), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9 (quat., CO) and 173.4 (quat., CO), m/z (FAB+) 630.2809 (MH+ C35H40N3O8 requires 630.2815).

Glycyl-L-2-methylprolyl-L-glutamic acid (L-2-MePE)

[00144] A mixture of protected tripeptide 7 (0.63 g, 1 0.00 mmol) and 1 palladium on activated carbon at 10% by weight (0.32 g, 0.30 mmol) in 91:9 methanol - water (22 cm3) was stirred under an atmosphere of hydrogen at room temperature, protected from light, for 23 h. The reaction mixture was filtered through a pad of Celite™ and the pad washed with 75:25 methanol - water (200 cm 3 ). The filtrate was concentrated to dryness under reduced pressure and the residue triturated with anhydrous diethyl ether to obtain a 38:1 mixture of G-2-MePE and tentatively methylamine 8 (0.27 g, 86%) as a white solid. very hygroscopic. Reversed phase HPLC analytical studies on the mixture [Si Altech Econosphere C18 column, 150 x 4.6 mm, 5 µm; 5 min. fluxed with H2O (0.05% TFA), then steady gradient for 25 minutes to MeCN as eluent at a flow rate of 1 mL/minute, diode array detection] indicated it was a 38:1 mixture of two elution peaks with retention times of 13.64 and 14.44 minutes at 207 and 197 nm, respectively. G-2-MePE was shown to be a 73:27 mixture of trans:cis conformers by 1H NMR analysis (ratio was estimated from relative intensities of doublet and triplet at δ 4.18 and 3.71, yielded to Gluα- protons. H of the major and minor conformers, respectively): melting point: 144°C; [α]D -52.4 (c 0.19 in H2O), δH (300 MHz, D2O; internal MeOH) 1.52 (3H, s, Proα-CH3), 1.81 to 2.21 (6H, m, Proβ-H2, ProY-H2 and Gluβ-H2 ), 2.34 (1.46 H, t, J 7.2, GIUY-H2), 2.42* (0.54H, t, J 7, 3, GIUY-H2), 3.50-3.66 (2H, m, Pro'-H2), 3.71* (0.27H, t, J 6.2, Gluα-H), 3.85 (1H , d, J 16.6, gIiα-HAHB), 3.92 (1H, d, J 16.6, gIyα-HAHB) and 4.18 (0.73H, dd, J 8.4 and 4.7, GIUα-H), (75 MHz, D2O; internal MeOH) 21.8 (CH3, Proα-CH3), 25.0 (CH2, ProY-C), 27.8* (CH2, Gluβ-C), 28, 8 (CH2, Gluβ-C), 32.9 (CH2, Gluy-C), 40.8 (CH2, Proβ-C) 42.7 (CH2, gliα-C), 49.5 (CH2, Proδ-C ), 56.0* (CH, Gluα-C), 56.4 (CH, Gluα-C), 69.8 (quat, Proα-C), 166.5 (quat., Gly-CO), 177, 3 (quat., Pro-CON), 179.2 (quat., Gluα-CO), 180.2* (quat., GluY-CO) and 180.6 (quat., GluY-CO): m/z (FAB+) 316.1508 (MH+. C13H22N3O6 requires 316.1509).

Example 3: In Vitro Neuroprotection

[00145] The therapeutic effects of GPE analogues were examined in a series of in vitro experiments to determine their effects on the neurogeneration of neuronal cells of different origin. The in vitro systems described herein are well established in the art and are known to be predictive of neuroprotective effects observed in vivo, including effects on humans suffering from neurodegenerative disorders.

Material and methods

[00146] The following experimental protocol followed guidelines approved by the Animal Ethics Committee of the University of Auckland.

Preparation of cortical astrocyte cultures for collection of metabolized cell culture supernatant

A cortical hemisphere of a 1-day-old rat was used and collected in 4 mL of DMEM. Trituration was carried out using a 5 ml glass pipette and an 18 gauge needle. The cell suspension was sieved through a 100 µm cell sieve and washed in 50 ml DMEM (centrifugation for 5 minutes at 250 g). The pellet was resuspended in 20 ml DMEM + 10% fetal calf serum. The suspension was added to two 25 cm3 flasks (10 ml per flask) and cultured at 37°C in the presence of 10% CO2 followed by a change of medium twice a week. When cells reached confluence, they were washed three times with PBS, adjusted to Neurobasal/B27 and incubated for an additional 3 days. This supernatant was frozen for temporary storage at -80°C.

Preparation of Stracial and Cortical Tissue from E18/E19 Mouse Embryos

[00148] A pregnant female rat was euthanized by CO2 treatment and then prepared for caesarean section. After surgery, the embryos were removed from their amniotic sacs and decapitated. The heads were placed on ice, in DMEM/F12 medium for striatum and PBS + 0.65% D(+) - glucose for cortex.

Striated Tissue Extraction Procedure and Cell Preparation

The entire brain was removed from the skull ventral side up in DMEM/F12 medium. The striatum was dissected from both hemispheres under a stereomicroscope and the striatal tissue was placed in a Falcon tube on ice. Striatal tissue was then minced using a PI000 pipette to 1 ml volume. The tissue was carefully minced by pipetting the solution up and down the pipette tip approximately 15 times, using shear force in alternate fluxes. The pieces of fabric settled to the bottom of the Falcon tube within 30 seconds. The supernatant containing a suspension of dissociated single cells was then transferred to a new sterile Falcon tube on ice. The tissue pieces were reground to avoid excessively damaging the already dissociated cells, by excessive grinding. One milliliter of ice-cold DMEM/F12 medium was added to the tissue pieces in the first tube and ground as before. The tissue pieces were seated and the supernatant removed to a new sterile Falcon tube on ice. Cells were centrifuged at 250g for 5 minutes at 4°C.

Placing on Plating and Cultivation of Striatal Cells

The striatal cells were plated in 96-well Poly-L-Lysine (0.1 mg/ml) plates (only the 60 internal wells) at a density of 200,000 cells/cm2 in Neurobasal/B27 medium (Invitrogen) . Cells were cultured in the presence of 5% CO2 at 37°C with 100% humidity. The medium was changed on days 1, 3 and 6.

Cortical Tissue Extraction Procedure and Cell Preparation

[00151] The two cortical hemispheres were carefully removed from the entire brain by a spatula, with the ventral side facing up in a petri dish containing PBS + 0.65% D(+)-glucose. Forceps were placed on the rostral (near the B.olfatorius) part of the cortex in order to fix the tissue and two lateral-sagittal oriented cuts were made to remove the paraform and entorhinal cortex. A frontal oriented cut at the posterior end was made to remove the hippocampal formation. A final frontal cut was made a few millimeters away from the last cut in order to obtain an area retention of 17/18 of the visual cortex.

[00152] Cortices were placed on ice in PBS 0.65% (+) - glucose and centrifuged at 350 g for 5 minutes. The supernatant was removed and trypsin/EDTA (0.05%/0.53 mm) was added for 8 minutes at 37°C. The reaction was stopped by the addition of an equal amount of DMEM and 10% fetal calf serum. The supernatant was removed by centrifugation, followed by two subsequent washes in Neurobasal/B27 medium.

The cells were minced once with a Pasteur glass pipette, in 1 ml of Neurobasal/B27 medium and subsequently twice, using a 1 ml insulin syringe with a 22 gauge needle. was passed through a 100 µm cell filter and washed in 1 ml of Neurobasal/B27 medium. Cells were counted and adjusted to 50,000 cells per 60 µL.

Placing in Plate and Cultivation of Cortical Cells

96-well plates were coated with 0.2 mg/ml poly-L-lysine and subsequently coated with 2 µg/ml laminin in PBS, after which 60 µl of cortical astrocyte conditioned medium was added to each well. Subsequently, 60 µL of cortical cell suspension was added. Cells were cultured in the presence of 10% CO2 at 37°C with 100% humidity. On day 1, there was a complete medium change (1:1 - Neurobasal/B27 and astrocyte conditioned medium) with the addition of 1 µM cytosine-β-D-arabinofuranoside (mitosis inhibitor). On days 2 and 5, 2/3 of the middle were changed.

P8 Animal Cerebellum Microexplants: Preparation, Cultivation and Fixation

[00155] Laminated cerebellum cortices from both hemispheres were explanted from a P8 mouse, cut into small pieces in a solution of PBS+ 0.65% D (+) glucose and ground with a 23 gauge needle and subsequently pressed through a 135 µm pore size sieve. The microexplants obtained were centrifuged (60 g) twice (mean variation) in STARTV medium supplemented with serum-free BSA (Biochrom). For cultivation, 40 μL of cell suspension was adhered for 3 hours on a slide with a sliding coating with 0.1 mg/mL of Poly-L-Lysine placed in six-well plates sized at 35 mm, in the presence of 5% of CO2 at 100% humidity at 34°C. Subsequently, 1 ml of STARTV medium was added along with the toxins and drugs. Cultures were monitored (evaluated) after 2-3 days of culture, in the presence of 5% CO2 under 100% humidity. For cell count analysis, cultures were fixed in paraform aldehyde at increasing concentrations of (0.4%, 1.2%, 3% and 4%, for 3 minutes each), followed by a wash in PBS.

Administration of Toxins and Drugs to Neural Cells In Vitro and Data Analysis

[00156] To study the neuroprotective effects of GPE analogs, we performed a series of in vitro experiments with okadaic acid to cause toxic damage to neural cells. Okadaic acid is an art-recognized toxin that is known to damage neurons. In addition, neural cell recovery or neural cell function after okadaic acid injury is recognized as predictive of recovery from injuries caused by other toxins.

[00157] To cause toxic damage to neurons, we exposed neurons to 1:100 parts of okadaic acid, in concentrations of 30 nM or 100 nM and 0.5 mM of 3-nitropropionic acid (only for microexplants of cerebellum) . GPE (1 nm and 1 mm), or G-2-MePE (1 NM-1 mM) was used 8 days in vitro (DIV) in cortical cultures and 9DIV for striatal cultures. Incubation time was 24 hours. Survival rate was determined by a colorimetric endpoint MTT assay at 595 nm in a multi-well plate reader. For cerebellar microexplants, four windows (field 0.65 mm2) with the highest cell density were chosen and cells showing neurite outgrowth were counted.

Results

[00158] The GPE analogue, G-2-MePE exhibited comparable neuroprotective effects in all three systems tested in vitro (figures 12-15).

[00159] Cortical cultures responded to concentrations of 10 µM of GPE (figure 12) or G-2-MePE (10 µM, figure 13) with 64% and 59% neuroprotection, respectively.

[00160] The other two types of cultures demonstrated neuroprotection at lower doses of G-2-(MePE (cerebellar microexplants: figure 14 and striatal cells: figure 15). Striatal cells demonstrated neuroprotection within the range of 1 nM and 1 mM of G-2-MePE (figure 15), whereas postnatal cerebellar microexplants demonstrated neuroprotection with G-2-MePE in the dose range between about 1 nm and about 100 nM (figure 14). G-2-MePE is a neuroprotective agent and may have therapeutic effects in humans suffering from neurodegeneral disorders Since G-2-MePE can be neuroprotective when administered directly to neurons in culture, G-2-MePE it can be effective in vivo when administered directly to the brain of affected animals.

Example 4: Effects of G-2-MePE on Striatal Cholinergic Neurons on Aging Rats

[00161] To determine whether G-2-MePE can affect cholinergic neurons, we studied the aging of rats. Choline acetyltransferase (ChAT) is an enzyme that is involved in the biosynthesis of the cholinergic nerve neurotransmitter acetylcholine. It is well known that ChAT immunodetection can be used to determine the number of cholinergic nerves present in a tissue. It is also known that the number of cholinergic nerves present is related to the physiological function of the cholinergic neural pathways in the brain.

[00162] In this experiment the effects of G-2-MePE on the number of ChAT-positive neurons in the brain of 18-month-old rats were tested.

Methods

Eighteen-month-old rats received a total of five treatments. One control group was treated with the vehicle (brine solution only (n = 4) and four groups were treated with a single dose of G-2-MePE. Doses of 0.012 (n = 4), 0.12 (n = 5), 1.2 (In = 5) and 12 mg/kg (n = 3), respectively, were injected subcutaneously.The rats were sacrificed with an overdose of pentobarbital 3 days after drug treatment. fused with normal brine solution and 4% paraform aldehyde and fixed in perfusion fixative overnight.Brains were stored in 25% sucrose in 0.1 M PBS (pH 7.4) until tissue sinks. Frozen coronal striatum were cut with a microtome and stored in 0.1% sodium azide in 0.1 M PBS at 4° C. Immunoreactivity for choline acetyltransferase (ChAt) was created by staining using a free-floating section method. Briefly, antibodies were diluted in 1% goat serum The sections were incubated in 0.2% triton in 0.1 M PBS/Triton™ at 4°C. overnight before immunohistochemical staining. Sections were pretreated with H2O2 1% to 50% methanol for 20 minutes. The sections were then incubated with rabbit anti-ChAT antibodies (Rb) (1:5000), (the primary antibodies) in 4D, on a shaker, for two days. Sections were washed using PBS/Triton™ (15 min 3d) and then incubated with biotinylated goat anti-rabbit secondary antibodies (1:1000) at room temperature overnight. Sections were washed and incubated in ExtrAvidin™ (Sigma) (1:1000) for 3 hours and followed by H2O2 (0.01%) in 3,3-diaminobenzene tetrahydrochloride (DAB, 0.05%) to produce a colored reaction product. These sections were mounted on coated chromium-alum slides, dried, dehydrated and covered.

[00164] The striatal neurons in both hemispheres exhibiting specific immunoreactivities corresponding to ChAT were counted using a light microscope and a 1 mm 2x1000 grid. The size of the striatal region used for counting was measured using an image analyzer. Total neuron counts/mm2 were compared between groups.

Data were analyzed using a paired t-test and presented as mean +/- SEM. The results are shown in Figure 16.

Results

[00166] Figure 16A shows that the number of ChAT-immunopositive neurons increased in the brains of animals treated with G-2-MePE. This clearly indicates that the administration of G-2-MePE is effective in increasing the ChAT level in the brains of aged rats. Since ChAT is an enzyme involved in the synthesis of the cholinergic neurotransmitter acetylcholine, we conclude that G-2-MePE can increase the amount of cholinergic transmitter in the brain of middle-aged rats.

Example 5: Effects of G-2-MePE on Spatial Reference Memory in Mice

[00167] Having demonstrated that G-2-MePE can increase ChAT and therefore has the potential to improve cholinergic neuronal function, we then examined whether G-2-MePE might be useful in treating cholinergic-related disorders. age in cognition and/or memory. Therefore, we carried out a series of studies in rats using well-established tests for memory.

Experiment 1: Morris Maze Water Test

[00168] The Morris Maze Aquatic Test is a well-recognized test for assessing spatial reference memory in rats.

individuals

[00169] We employ 12, 8 or 4 month old Wistar rats.

Methods Test environment and devices

[00170] The Morris Maze Aquatic Test was performed using a black plastic pool filled to a depth of 25 cm in height with black colored water with a non-toxic dye. The pool had a circular black insert so the walls also appeared uniformly black. The pool was divided into four quadrants (north, south, east and west) by two imaginary perpendicular lines that crossed in the center of the pool. A metallic platform was placed in the geographic center of the SE quadrant 50 centimeters from the edge of the pool so that it was 2 cm below the water surface and invisible. The platform remained in that position through training.

[00171] The experiment used extra maze clues (ie objects in the environment around the pool) so the rats could use to navigate to the platform. Distinct posters or paintings were hung on the walls. Furniture in the environment was not moved during the testing period. The placement of the pool allowed the experimenter to have easy access to it from all sides. The pool was emptied and renewed daily during the tests, with water at 25°C +/- 2°C.

[00172] The farthest point in the pool (in relation to the experimenter's position) was designated as "north", and the other cardinal points "east", "south" and "west" were the rightmost points, point bottom and leftmost pool, respectively.

These points were marked with duct tape on the outside of the pool. acquisition phase

[00173] The rats in each group were trained to swim to the submerged platform. Rats received six 60-second trials per day for four consecutive days. The experiment began by placing the mouse in the water facing the pool wall at one of the four departure locations (north, south, east, west). The sequence of starting locations was chosen pseudonymously, so that the starting location of a given experiment was different from that of the previous experiment, and no starting location was used more than twice during daily training. The same sequence of locations was used for all rats on a given day, but it varied between days. The experiment ended when the mouse found the platform, or within 60 seconds, which has never happened before. The trials were timed with a stopwatch. If the mouse found the platform, it could remain there for 15 seconds before being removed to a storage container. If the platform was not found, the mouse was guided to it manually and placed on the platform for 15 seconds. The interval between experiments was 60 seconds. The storage container was covered in order to minimize any interference between experiments. After completing the daily tests by a rat, the animal was dried with a towel and placed under the heat lamp in the storage basket until its fur was dry. The time needed to find the platform (latency time, seconds) was obtained for each mouse in each experiment. If the mouse did not find the platform in a given test, its latency score was the maximum duration of that experiment (60 seconds).

drug treatment

[00174] Three days after completion of the acquisition phase, osmotic mini-pumps (Alzet) were implanted subcutaneously under halothane anesthesia) to dispense drug or vehicle continuously for 1 or 3 weeks. After completion of the perfusion, the pumps were removed and the wounds were sutured again.

[00175] The five treatment groups were: 1. brine one week (n was originally 7, but one rat that lost weight quickly was excluded and later found to have a pituitary tumor); 2. 3 weeks brine (n=8); 3. G-2-MePE low dose (0.96 mg/day) 1 week (n = 8); 4. G-2-MePE low dose (0.96 mg/day) 3 weeks (n = 8); 5. G-2-MePE high dose (4.8 mg/day) 3 weeks (n = 7).

Four (n = 3) and eight month old (n = 9) control rats received no drug treatment. The 12-month-old rats were assigned to one of five groups based on their swim times relative to acquisition, such that the groups were approximately equivalent in their average performance prior to receiving any medication.

Retention Phase (Reference Memory)

[00177] The rats' ability to remember or relearn the original location of the platform was tested four weeks after the original training. This means that residual medicine could have been withdrawn for a minimum of 7 days in the case of the 3-week pumps and 21 days in the case of the 1-week pumps. The retention test procedure was identical to the acquisition. Pharmacokinetic studies indicate that the plasma concentration of subcutaneously administered G-2-MePE rose to a peak and then declined with a first-order kinetic pattern with a plasma half-life (t[1/2] ]) between about 30 and 60 minutes. Thus, by the time the retention study was carried out, at least 7 days after the removal of the minipumps containing G-2-MePE, almost all of the G-2-MePE had been eliminated from the animals' circulation.

Data analysis

[00178] Swimming latency for each rat was recorded for each experiment for each day of the acquisition and retention phase and changes between phases were examined by Analysis of Variance.

[00179] The 3-week vehicle and 3-week high-dose G-2-MePE were compared in acquisition and retention. The high dose of G-2-MePE given for 3 weeks improved retention of the original water maze task after an interval of 4 weeks.

Results

[00180] Figure 17 shows the comparison between elderly rats treated with high dose G-2-MePE (4.8 mg/day) and low doses (0.96 mg/day), and aged rats treated with brine solution , with the young controls (4 months) used as controls. Before treatment with G-2-MePE, no differences were observed between the elderly groups (12 months of age). In contrast, 4-month-old animals needed less time to reach the platform than older animals. After a period of 3 weeks without testing, during which time both the brine and G-2-MePE were administered, the animals that received only the brine showed no improved ability to reach the platform, as indicated by the similar times needed on the fourth day of the test of the acquisition phase and first day of the retention phase. In contrast, animals that received G-2-MePE treatment at either high or low dose had improved memory as reflected in a decrease in the time taken to reach the platform relative to controls treated with brine solution. Furthermore, animals treated with G-2-MePE performed similarly to 4-month-old young animals (figure 17) and 8-month-old animals (data not shown). Thus, it is concluded that G-2-MePE can improve memory in middle-aged rats that had previously shown memory deficits compared to young rats. Furthermore, since at the time of the retest, the G-2-MePE had been withdrawn from circulation, we can conclude that the memory-enhancing effects of G-2-MePE were likely due to the improved function of cholinergic neurons.

Experiment 2: 8-Arm Radial Maze Test

[00181] Five months after the initial experiment, rats now 17 months old were retested for spatial working memory in a radial arm maze.

Methods Device

[00182] The apparatus consists of a central platform communicating with 8 identical arms, each with a cup of food at the end of the arm.

test procedure

[00183] The rats were partially deprived of food for at least 10 days before and throughout the radial maze procedure.

[00184] The maze was assembled and positioned so that the experimenter could clearly observe the behavior of the rats from a predetermined location. The experimenter numbered the arms of the maze according to their orientation from one to eight in a clockwise direction.

Pre-Training (Pre-Medication)

[00185] On day one the ports were inserted into the arms and each rat was confined to the central platform with 20 microspheres of feed for 5 minutes. This continued once a day for four days, and all rats were observed consuming some microspheres. The next day, the rats were allowed to explore the maze for five minutes. All arms received two microspheres of feed in the food cup located at the end of each arm, and one microsphere at both the entrance and the middle of each arm. This was repeated for at least five, but up to eight days for rats that explored fewer than eight arms in two consecutive sections. All rats had a final section on the ninth day of pretreatment. At this point it was decided that one of the elderly rats that had only made one entry into the arm on eight out of nine days would be excluded from further testing of this procedure. Otherwise, all rats were included, regardless of how much exploration they performed pre-training.

[00186] There was no statistically significant difference between the elderly groups in the number of arms visited at the end of the pre-training section (Drug: F (2.31) = 0.44, p = 0.65).

drug treatment

Thirty days before testing (five days after pre-training) 17-month-old male Wistar rats were implanted (under halothane anesthesia) with subcutaneous osmotic mini-pumps (Alzet) to dispense medication continuously for 3 weeks. After completion of the perfusion pumps were removed and wounds resutured (9 days with no drug ingestion being allowed).

[00188] The treatment groups were as follows: 1. young controls (4 months), n = 6; 2. brine n=10; 3. G-2-MePE low dose (2.4 mg/kg/day) n = 13 4. G-2-MePE high dose (12.4 mg/kg/day) n = 5

[00189] Brine and low-dose groups are composed of all rats that received treatments in phase 1 of the experiment (when rats were 12 months old), regardless of whether they had one or three weeks of treatment. One mouse in each of the high dose and brine groups was removed due to skin tumors. One of the low-dose rats did not participate in this experiment, due to the fact that it cannot be pre-trained (see below).

Tests (Post-Medicine)

[00190] The working memory tests began on the ninth day of the drug-free period. Rats received 10 training sessions per day for 12 days. The procedure was the same as for pre-training, but only the food cups were placed as bait. The rats were given six minutes to make up to 16 choices, visiting any of the eight arms. Choice was defined as occurring when all four legs were within one arm. The researcher recorded the sequence of entries in the arms with pen and paper. Sessions were terminated after all eight arms had been entered, 16 choices made, or 6 minutes had elapsed. The time taken to enter all eight arms, when it occurred, was recorded.

Data analysis

[00191] An arm choice was considered correct when the rat enters a previously unvisited arm. Performance was rated daily according to the following parameters: 1) Correct choice (CC) 8-12 is the number of correct choices made divided by the total number of choices made. For animals that failed to visit all eight arms in a trial, n denominator of the ratio is considered to be 12. 2) Working Right Choice (WCC) 8-12 is the measure from which the memory data of work are derived. Data were collected as described for CC 8-12 above, but for this parameter, only rats that entered all 8 arms in one session were included.

[00192] Rats that made less than 8 entries in the arms were not used to checking working memory because they could not remember which arms they had visited previously and therefore had such impaired memory that they were unable to complete the test. of the animals that, for whatever reason, did not explore the labyrinth.

Results

[00193] CC8-12: There was an overall improvement of all groups between 10 days (F (9.324) = 4.01, p < 0.0001), but no significant effect in the group (F (3.36) = 0.19 , ns) or Group X Days of Interaction (F (27,324) = 1.05, ns) (data not shown)

[00194] WCC8-12: Figure 18A shows the acquisition profile according to the WCC8-12 score between the 10 days of testing. There was a significant effect of Group (F (3, 12) = 4.27, p = 0.029) and Days (F (9, 108) = 2.09, p = 0.036), but the interaction between these factors was not significant (F ( 27.108 ) = 1.06, ns). The high dose G-2Me-PE group showed the greatest improvement between days, followed by the young controls. There was very little difference between low dose G-2Me-PE and brine solution.

[00195] Figure 18B shows that the results indicate that rats exposed to the highest dose of G-2-MePE (n = 5) had made more correct entries to obtain feed microspheres compared to vehicle-treated rats (* p<0.05, n=10). It is concluded from this study that G-2-MePE improves spatial memory in elderly rats.

Example 6: G-2-MePE increases neuroblast proliferation and decreases astrocytosis in the brains of aged rats

[00196] Since neuronal degeneration can result in decreased number of neurons, a desirable therapeutic goal is to increase the number of neurons in the brain. Neurons are derived from neuroblasts, a cell less differentiated than a neuron but within the neural lineage. Typically, a neuroblast is exposed to conditions that cause it to mature into a mature phenotype, with a defined sum of neural processes (axons and dendrites) and finally making connections with other neurons (eg, synapses). Thus, measuring neuroblast proliferation has become a well-known early marker for nerve cell proliferation. Thus, detecting an increase in neuroblast proliferation induced by a pharmaceutical agent is an accepted method for predicting neural cell growth in animals. Since rats and humans share similar mechanisms of neural cell proliferation, detection of changes in neuroblast proliferation in rats in vivo is predictive of similar effects in humans.

[00197] It is also known that a histological correlation of impaired cognitive function is an increase in the number of astrocyte cells in the brain of affected animals. So, to determine whether G-2-MePE might be useful in stimulating neuroblast proliferation and in treating astrocytosis, we performed a series of studies in elderly rats.

Methods and Materials immunohistochemistry

[00198] To perform these studies, tissues were fixed and embedded in paraffin and cuts obtained using conventional methods. Coronal sections (6 μm) containing the level of the hippocampus were cut and mounted on chromium-alum-coated slides for staining. The sections were dewaxed in xylene, dehydrated in ethanol series and incubated in 0.1 M phosphate buffered saline (PBS).

[00199] Primary antibodies against glial fibrillary acidic protein (GFAP) and on proliferating cell nuclear antigen (PCNA) have been used to label reactive glial cells and cells undergoing apoptosis and proliferation, respectively. For antigen unmasking (caspase-3 and PCNA staining), sections were heated in 10 mM sodium citrate buffer (pH 6.0) for 1 minute at high power. All sections were pretreated with 1% H2O2 in 50% methanol for 30 min. to saturate endogenous peroxidase activity. Then 1.5% normal horse serum or 2.5% normal sheep serum in PBS was applied for 1 hour at room temperature to block non-specific background staining. The sections were then incubated with the following primary antibodies: anti-GFAP monoclonal antibody (Sigma, St. Louis, MO, USA diluted 1:500); mouse anti-PCNA antibody (DAKA, A/S, Denmark, diluted 1:100). After incubation with the primary antibodies at 4°C for 2 days (except for PCNA staining which was incubated overnight), the sections were incubated with either the biotinylated horse anti-mouse antibody or the secondary goat anti-rabbit antibody (1:200, Sigma) at 4°C overnight. ExtrAvidin™ (Sigma, 1:200), which had been prepared 1 h before its use, was applied for 3 hours at room temperature, and then reacted in 0.05% 3,3-diaminobenzidine (DAB ) and PBS to produce a brown reaction product. Sections were dehydrated in a series of alcohols in xylene and placed on slide-coated glass slides with mounting medium.

[00200] Immunohistochemical staining was performed on brain samples taken from both the control and G-2-MePE treated groups of young rats (4 months old), or middle-aged rats (9 months old) and elderly (18 months).

[00201] Control sections were treated in the same manner except that the primary antibody was omitted from the incubation solution. The number of PCNA positive cells was counted in the subventricular zone and GFAP positive cells were labeled in the cerebral cortex.

Experiment 1: G-2-MePE stimulates the proliferation of neuroblasts in the brain of aged rats

[00202] The subventricular zone (SVZ) and the dentate gyrus (DG) are two regions of the brain hosting adult neurogenesis. Decreased neurogenesis in both SVZ and DG has been well reported to be correlated with memory decline with aging and effects of nerve growth factor and epidermal growth factor and memory enhancement being reported to be due to increase in progenitor SVZ proliferation. The use of PCNA as a cell proliferation marker, cell proliferation in SVZ was examined by counting the number of cells that are PCNA positive. In selected animals, at least some of the proliferating cells were identified as neuroblasts, as stained with the neural cell-specific agent, doublecortin.

Eighteen month old male rats were treated intra-peritoneally with a single dose of G2-MePE (doses of 0, 0.012, 0.12. 1.2, 12 mg/kg). Brains were harvested 3 days after treatments and immunohistochemical staining of PCNA and GFAP was performed. The number of PCNA positive cells was counted in the SVZ and the cell number was then calculated as cells/mm depending on the length of the ventricle wall used for counting (Fig. 19A). The group treated with the highest dose (12 mg/kg, n = 5) showed a significant increase in the number of PCNA positive cells compared to the group treated with vehicle (* p < 0.05, n = 7) . The data indicated a dose-dependent effect of G-2PE in enhancing neurogenesis.

[00204] Double label fluorescence indicated co-localization of PCNA with doublecortin label employed in neuroblasts. Figure 19B is a photograph of a portion of the rat brain exhibiting an increase in both PCNA (green, 20-fold) and doublecortin (red, 20-fold) in rats treated with the highest dose of G-2-MePE (right panel ) compared to vehicle treated rats (left panel). The two markers were clearly co-located (figure 19B, photo, 100 times). We conclude that G-2-MePE can stimulate the proliferation of brain cells, including neuroblasts. Since neuroblasts are precursor cells of neurons, we can further conclude that G-2-MePE can increase the population of neurons in the brains of animals treated with the compound of this invention.

Experiment 2: G-2-MePE stimulates neuroblast proliferation in middle-aged rat brain ZSV

[00205] Effects of G-2-MePE (1.2 mg/kg) were studied in a group of middle-aged nine-month-old rats. L-2-MePE (1.2 mg/kg) or vehicle was administered intraperitoneally (i.p.). Cell proliferation in the SVZ was examined three days after treatment using PCNA immunohistochemical staining. Fig. 19C shows a significant increase in the number of PCNA positive cells after G-2-MePE treatment (** p < 0.005, n = 4). As some of the proliferating cells stained with PCNA were identified as neuroblasts (see Experiment 1 above), we can conclude that G-2-MePE can stimulate neuroblast proliferation in middle-aged rat brains.

Experiment 3: Astrocytosis in Aging Brains

[00206] Growing evidence suggests that dysfunction of astrocytes in old age can provoke inflammation, leading to further neuronal degeneration. Upregulation of activated astrocytes has been well reported and is closely associated with memory decline with aging, perhaps through depression in endogenous neurogenesis.

[00207] Using GFAP as a marker for reactive astrocytes, the number of GFAP-positive cells was counted in the hippocampal subregion of aged rats treated with G-2MeP or vehicle. We found a significant increase in reactive astrocytes in the hippocampus of elderly animals (figure 20A), and in the cerebral cortex. Some of the astrocytes were associated with capillaries (pictured in figure 20B arrows) in aged rats compared to both young rats (*p<0.01) and in middle aged rats (*#p<0.01).

[00208] As part of the vascular component, GFAP positive astrocytes also play a role in angiogenesis (figure 20B, arrows), which also contribute to the inflammatory response in the brain. Therefore, the elevated GFAP astrocytes seen in aging brains may indicate a chronic stage of brain degeneration.

Experiment 4: G-2-MePE Reduces Astrocytosis in Aged Brains

The effects of G-2-MePE on astrocytosis in the CA4 subregion of the hippocampus in aged rats have also been evaluated. 18-month-old male Wistar rats were divided into five treatment groups as follows: vehicle, 0.12 mg/kg/day, 0.12, 1.2, and 1.2 mg/kg/day (n = 6 each).

[00210] GFAP-positive cells were counted using a computer program (Discovery 1). The results are shown in figures 20C and 20D. G-2-MePE was administered intraperitoneally and the number of GFAP-positive cells was assessed 3d after injection. Using a visual scoring system (0 = no astrocytes, 1 = few astrocytes, 2 < 50%, 3 > 50%) he estimated the number of astrocytes in five different cortical regions.

[00211] Treatment with G-2-MePE reduced the number of reactive astrocytes in the CA4 region of the hippocampus compared to the vehicle-treated group (figure 20C, * p < 0.05), especially the groups treated with doses of 0.12 and 12 mg/kg. A similar effect was seen for G-2-MePE in the cerebral cortex (figure 20D).

[00212] There are usually few GFAP positive astrocytes located in the deep layer of the cortex of the rat brain and those that are present are usually in close association with the white matter bands. However, we found that there were GFAP-positive cells in the middle layer of the cortex, intimately associated with blood vessels.

[00213] The results of the studies presented in this document indicate that aging is associated with several changes in the brain. First, there is an age-dependent loss of memory and cognitive function. Second, there is an age-dependent increase in astrocytes. All of these findings in rats are consistent with other and known roles of cholinergic nerves in maintaining cognitive function and memory in experimental animals and in humans.

[00214] We have found, unexpectedly, that a GPE analogue, G-2-MePE, released to aged animals reverses, at least partially, all of the preceding age-associated changes. First, G-2-MePE increases the amount of ChAT present in the brain cells of animals exposed to neurotoxins, okadaic acid or 3-NP. This effect of G-2-MePE mimicked a well-known neuroprotective agent, GPE. These effects were seen in cortical cells, cerebellum cells and striatal cells, indicating that the effects were widespread in different portions of the brain. Second, G-2-MePE increased the amount of ChAT in the striatum, indicating that cholinergic neurons are sensitive to G-2-MePE. These observed chemical and histological changes were accompanied by behavioral changes. Elderly animals treated with G-2-MePE exhibited improved memory in two well-known test systems compared to vehicle-treated controls. Then, G-2-MePE induced neuroblast proliferation in aged brains. Finally, treatment with G-2-MePE reversed the increase in astrocytosis seen in the hippocampus and cortex of aging brains. The effects of G-2 -MePE were not due to the acute effects of the agent; because in many of the studies cited in this document, sufficient time had elapsed from the cessation of drug release for the trial, with little or no drug present.

Example 7: Comparison between the pharmacokinetics of GPE and G-2-MePE

[00215] The purpose of these studies was to compare the pharmacokinetic profiles of GPE and G-2-MePE in animals in vivo using conventional pharmacokinetic methods.

Methods

[00216] Adult male Wistar rats weighing between 180 and 240 g were used to determine the pharmacokinetics of GPE and G-2-MePE. To facilitate intravenous bolus injections and blood sampling, rats were implanted with an indwelling jugular cannula under halothane anesthesia three days prior to the experiment. Groups of six rats received a single intravenous bolus injection of either 30 mg/kg GPE or 10 mg/kg G-2-MePE dissolved in 0.1 M succinate buffer (pH 6.5). Blood samples (approximately 220 µL each) were collected into heparinized tubes containing Sigma mammalian tissue protease inhibitor cocktail within 0 to 10 min. prior to injection of any GPE or G-2-MePE, and 1, 2, 4, 8, 16, 32, 64, and 128 minutes after injection of any GPE or G-2-MePE. Samples were centrifuged at 3000g for 15 min. at 4°C and the plasma removed and stored at -80°C until extraction and assay by either radioimmunoassay ("RIA") or reverse-phase HPLC. The RIA and HPLC methods used were the conventional ones.

[00217] Elimination of the drug after a single intravenous bolus injection was verified as a first-order process, following the equation C = Coe-kt where C represents the concentration of the drug, at any point of time, Co represents the concentration when time (t) is equal to zero and k is the first-order rate constant, expressed in units of concentration per hour. The half-life ke (t1/2) were calculated from the slope of the linear regression line in the elimination phase of the semi-log plot of plasma concentration versus time as: Log C = -kt/2.3 + log Co Results were expressed as mean ± standard deviation.

Results

[00218] Figure 21 shows a graph of the in vivo plasma concentrations of GPE2 and G-MePE after intravenous (iv) injection. Filled squares represent the concentrations of GPE at each time point, and filled triangles represent the concentrations of G-2-MePE at each time point.

[00219] The plasma concentrations of GPE and G-2--MePE were markedly increased within one minute after injection. After injection of 30 mg kg of GPE, a peak concentration of 40.0 ± 10.8 mg/mL was observed. Plasma GPE concentrations then rapidly decreased according to a first-order kinetics process. The first order rate constant for the GPE was found to be 0.15 ± 0.014 ng/mL/min., t1/2 was found to be 4.95 ± 0.43 min. and the estimated clearance of GPE from plasma was found to be 137.5 ± 12.3 mL/hour.

[00220] After injection of 10 mg/kg of G-2-MePE, the peak concentration was found to be 191 ± 16.1 mg/ml. Plasma concentrations of G-2-MePE then declined according to a first-order kinetics process. The first order rate constant for G-2-MePE was found to be 0.033 ± 0.001 ng/mL/min., t % was found to be 20.7 ± 0.35 min. and the estimated clearance was 30.1 ± 0.5 mL/hour.

[00221] After injection, the maximum plasma concentration of G-2-MePE was about 4.8 times greater than the maximum plasma concentration of GPE, despite the higher dose of GPE delivered (30 mg/kg) compared to dose of G-2-MePE released (10 mg/kg).

The discovery of higher plasma concentrations of G-2-MePE relative to GPE at all time points less than 125 minutes, despite a lower release dose of G-2-MePE, was totally unexpected. based on known plasma concentrations of GPE. The t% for G-2-MePE was more than 4 times longer than the t1/2 for GPE.

[00223] The finding of a longer halfway of G-2-MePE compared to that of GPE was completely unexpected based on t % GPE. The increased t % of G-2-MePE means that G-2-MePE is clearly slower from circulation. This check is totally unexpected based on the GPE debug rate.

[00224] We conclude from these studies that G-2-MePE is a potent agent capable of reversing many of the adverse effects of aging on the brains of animals, including humans. GPE analogues, including G-2-MePE therefore, may produce desired therapeutic effects, including neuroprotection, memory enhancement, increased neuroblast proliferation, and reduction of astrocytosis, and may be useful in reversing or attenuating the adverse effects of aging in humans.

While the present invention has been described in terms of certain preferred embodiments, it will be apparent to one of ordinary skill in the art in light of the knowledge and this disclosure that equivalents of the compound of the present invention can be prepared and administered under the conditions described. in the present application, and all such equivalents are intended for inclusion within the scope of the claims of the present application.

Example 8: Treatment of Rett Syndrome 1 Effects of G-2-MePE on Shelf Life and Long-Term Enhancement in the Rett Syndrome Model (RTT)

[00226] To determine whether G-2-MePE treatment might impact the development and progression of Rett syndrome, in a murine model of the disorder, male hemizygous MeCP2 mice (1 lox) were used. The MeCP2 knockout mouse system (MeCP2-KO) is widely accepted in the art as closely mimicking the range and severity of features of the neurological abnormalities of the human disorder, Rett syndrome.

[00227] All experiments were performed at the University of Texas Southwestern Medical Center and approved by the University of Texas Southwestern Medical Center Animal Care and Use Committee. G-2-MePE was synthesized at Al-bany Molecular Research Inc. (Albany, NY) and supplied by Neuren Pharmaceuti-cals Limited.

Methods Treatment

[00228] We treated male MeCP2 hemizygous mice (1 lox) with 20 mg/kg/day of G-2-MePE or brine solution (0.01% BSA, n = 15 per group in the survival experiment and n = 20 in the LTP experiment). Treatments were administered intraperitoneally from 4 weeks after birth. For the survival experiments, treatment was maintained during the course of the experiment. For the LTP experiment the mice were treated until the 9th week when they were used for slide preparation.

Survival

[00229] MeCP2-deficient mutant mice develop RTT systems at about 4-6 weeks of age and die between 10-12 weeks (Chen et al. De 2001. Nat Genet 27: 327-331). Survival of wild-type controls and MeCP2-deficient animals in vehicle and G-2-MePE treated groups was compared. Survival was measured weekly from the start of treatment (4 weeks) and used to generate survival curves for Kaplan-Meier that show the proportion of mice that survived (Y-axis) at each weekly interval (x-axis) (see Figure 22.).

Long Term Potentiation (Electrophysiology)

[00230] MeCP2-deficient mice have previously been reported to suffer from functional and ultrastructural synaptic dysfunctions, significant hippocampal-dependent memory impairment, and long-term hippocampal potentiation (LTP) (Moretti et al. The Journal of Neuroscience. 2006. 26. 26). (1):319-327). To test the effects of G-2-MePE treatment on synaptic function in the RTT model, we compared LTP from the hippocampus in both vehicle and 9-week-old G-2-MePE-treated animals. To do this, we measured the slope of fEPSP as a percentage of baseline neuron potential in hippocampal slices from MeCP2-deficient mice treated with either brine or G-2-MePE (figure 23).

Results

[00231] Figure 22 shows that treatment with G-2-MePE increased the survival of MeCP2 deficient mice. Wild type mice (top row) are control animals and therefore their survival was 100% at each time point. MeCP2-deficient mice treated with brine only died much more quickly (dotted line) compared to wild-type mice, such that about 11 weeks, only 50% of the MeCP-deficient mice survived. In stark contrast, however, we unexpectedly found that MeCP2-deficient mice treated with G-2-MePE survived much longer than mice treated with brine. In about 15 weeks, 50% of the animals survived. The data presented initially showed that MeCP2 mice were affected in terms of survival such that 50% of the animals died within 11 weeks in the untreated case. Animals treated with G-2-MePE showed improved survival, with 50 percent dying within 16 weeks. In this study, longevity data were compromised by inconsistent veterinary procedures, such that the mice did not have their teeth consistently serrated - a requirement in the MeCP2 mice not recognized at the beginning of the experiment. One consequence was the observation of early animal deaths unrelated to Rett Syndrome (particularly in the control group). A reexamination of the data showed that the effect of G-2-MePE persisted when the control group was operated on again, although the difference between groups was smaller (time to 50% death, 13.5 weeks in controls, 16 weeks in animals treated with G-2-MePE). No safety issues were raised by treating MECP2 mice with G-2-MePE.

[00232] These results demonstrated that G-2-MePE can substantially increase the survival of MeCP2 deficient mice. Since MeCP2-deficient mice can predict pathology and therapeutic efficacy in humans with Rett Syndrome, we conclude that G-2-MePE can increase life expectancy in humans with Rett syndrome.

[00233] Figure 23 shows the results of our studies to determine whether treatment with G-2-MePE increased long-term hippocampal potentiation (LTP) as measured by the fEPSP MeCP2 slope in deficient animals compared to treated mutant mice with brine solution. As shown in Figure 23, we unexpectedly found that G-2-MePE increased the fESPS slope in MeCP2 deficient mice compared to animals treated with only brine solution.

[00234] These results demonstrate that G-2-MePE can be effective in treating MeCP2-deficient mice in vivo. Since MeCP2-deficient mice are predictive of pathology and therapeutic efficacy in humans with Rett syndrome, we conclude that G-2-MePE may be an effective therapy for people with Rett syndrome.

Example 9: G-2-MePE Improves Dendritic Trees and Increases Dendritic Spine Length

[00235] We evaluated the effects of G-2-MePE treatment on dendrites. Transgenic mecp2 knockout mice (n = 15 to 20) were administered intraperitoneally with G-2-MePE at a dose of 20 mg/kg once daily. After sacrifice, dendritic spine density, spine length, and arborization were examined after Golgi staining after nine weeks, according to Table 1 below: Table 1: Sample size for all morphological analyzes of neurons and spine

[00236] Dendritic length was evaluated by the distance of the sum of a representative of hippocampal CA1 neurons from a male null mutant mouse in 9-week-old mecp2 treated with brine (3 neurons analyzed from three mice separately, n = 9) or G-2-MePE (20 mg/kg ip 1/day, from week 4 onwards; 3 neurons analyzed from 3 separate mice, n = 9).

[00237] We observed that G-2-MePE improved dendritic arborization and increased dendritic spine length. Figure 24 illustrates the results of this study. Dendritic length in µm (vertical axis) is registered as a function of distance (in µm; horizontal axis) from the sum of cells. For cells with narrow dendrites close to the cell body (sums), the dendrites were shorter. However, as the soma distance increased the brine treatment (open squares) produced dendritic lengths that increased to a maximum at a distance of 70 µm from the brine and declined at distances outside the soma. In contrast, treatment with G-2MePE (solid squares) produced longer dendrites over much of the soma distance range.

Example 10: Treatment of Rett Syndrome in Mice II Mice Mating and Genotyping

[00238] MeCP2 germline null allele mice are used (Chen et al., 2001). Genotyping is performed as in Chen et al. (Chen et al. 2001).

G-2-MePE Treatment

[00239] For survival measurements, nocturnal activity analysis and immunoblot analysis, G-2-MePE (synthesized Albany Molecular Research Inc. (Albany, NY) and provided by Neuren Pharmaceuticals Limited) is administered daily via intraperitoneal injections (20 mg/kg, vehicle = brine, 0.01% BSA). Treatment starts at PI 5 and is continued throughout the course of the experiments. For intracellular physiology experiments, mice are injected daily with G-2-MePE (20 mg/kg body weight, vehicle = brine, 0.01% BSA) for 2 weeks from P15 to P28, P32 , when they are used for the preparation of slides. For the optical imaging experiments, mice are injected with G-2-MePE (20 mg/kg body weight, vehicle = brine, 0.01% BSA) daily from the day of the cap suture for the day of the imaging.

Blade Physiology Preparation

[00240] Coronal sections (300 µm thick) in or near the sensorimotor cortex are cut at < 4°C ACSF using Vibratome. The slides were incubated at 37°C for 20 minutes after cutting, and at room temperature for the remainder of the experiment. The slides are transferred to a Warner chamber and recordings are made of pyramidal neurons located visually and identified in layer 5. Artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 25 mM NaHCO3, 1 mM NaHPO4, 3 mM KCl , 2 mM MgSO4, 2 mM CaCl2 and 14 mM glucose, are adjusted to 315-320 mOsm and pH 7.4, and bubbled with 95% O2/5% CO2. The intracellular pipette solution contained 100 mM potassium gluconate, 20 mM KCl, 10 mM HEPES, 4 mM MgATP, 0.3 mM NaGTP, and 10 mM Na-phosphate-creatine.

Whole Cell Intracellular Recordings

[00241] Borosilicate pipettes (3-5 MQ, WPI) are pulled through a Sutter P-80 puller (Sutter Instruments). Cells are visualized with a 40x water immersion Achroplan lens with DIC infrared lens (Zeiss) and detected with an infrared camera (Hamamatsu) projecting to a video monitor. Experiments are conducted by custom acquisition and real-time analysis software written by Matlab (Mathworks, Natick, Massachusetts) using a Multiclamp 700B amplifier (Axon Instruments) connected to a BNC 2110 connector block and M Series dual-channel acquisition card (National Instruments). Gigaseal and rupture is achieved and the integral cell registers are continuously checked for low levels of leakage and series resistance. For each record, a test pulse of 5 mV is applied to voltage clamp ~10 times to measure input and series resistance. Then, at clamp leakage current ~10 pulses (500 ms, 40-140 pA in 10 pA increments), are applied to quantify evoked firing rates and cell excitability. Access resistance, leakage, and intrinsic cell excitability are checked to be consistent across groups. Finally, spontaneous EPSCs under leakage current clamps at -60 mV are sampled at 10 kHz and passed through a low pass filter at 1 kHz. Analysis is performed using a custom software package written in Matlab, with all events detected according to automated limits and blindly checked for each event individually by the experimenter.

Golgi coloring

[00242] Samples (<1 cm) from P28 mice are fixed in 10% formalin and 2% silver nitrate for 2 days in the dark at room temperature. Sections of these samples are then cut to 50 µm thick in distilled water. The sections corresponding to the motor cortex are mounted on slides, air dried for 10 minutes, then dehydrated by sequential washes of 95% alcohol, 100% alcohol and xylene, and then sealed with a slip-coated slide. Images are obtained at 10x (whole cell) and 100x (spine imaging) using a Zeis Pascal 5 Exciter confocal microscope.

Optical imaging of intrinsic signals

[00243] Adult (> P60) wild type (SVEV or BL6) and MeCP2 (+/-) mutant females (BL6) are used for this experiment. The wild-type control group is composed of either wild-type litter of MeCP2 +/- females or matched SVEV wild-type or aged females. For monocular deprivation, animals are anesthetized with Avertin (0.016 mL/g) and the eyelid is sutured for 4 days. Before imaging, the suture is removed and the closed eye reopened. Only animals in which the deprivation sutures are intact and the deprived eye condition appears healthy are used for the imaging session. For activation of G-2-MePE signaling, a solution containing G-2-MePE is injected intraperitoneally (IP) daily during the entire deprivation period. For imaging sessions, mice are anesthetized with urethane (1.5 g/kg, 20% of the total dose is administered IP every 20-30 minutes until approximately the final dose, 0.02 mL of 1% chloroprothixene as well is injected together with the first administration). The skull is exposed and a custom-made plate is glued to the head to minimize movement. The skull is reduced on VI with a Dremel bur and covered with a solution of agarose in brine (1.5% and a glass slide with a sliding coating. During the imaging session, the animal is constantly oxygenated, its temperature is maintained with a heating blanket and eyes periodically treated with silicone oil, physiological conditions being constantly monitored. The anesthetized mouse is placed in front of a monitor that displays a periodic stimulus presented to both eyes, monocularly; the stimulus consisted of one white vertical or horizontal steering bar of 9° x 72° dimensions, 9 sec./cycle direction, on a uniformly gray background. The skull surface is illuminated with a red light (630 nm) and the The luminance tion is captured by a CCD camera (Cascade 512B, Roper Scienti-fic) at a rate of 15 frames/sec during each 25 minute stimulus session. A temporal high-pass filter (135 frames) is employed. o to remove slow signal noise, after which the signal is processed by the computer in order to extract, in each pixel, the Fast Fourier Transform (FFT) component corresponding to the stimulus frequency. The FFT amplitude is used to measure the resistance of the visual evoked response for each eye. The ocular dominance index is derived from each eye's response (R) at each pixel as ODI = (Ragainst-Ripsi)/(Ragainst + Ripsi). The binocular zone is defined as the region activated by stimulation from the ipsilateral eye to the imaged hemisphere.

Heart Rate Measurements

[00244] Real-time heart rate is measured using a tail clip sensor (Mouse OX Oximeter - Oakmont, PA). The mice are not anesthetized but physically contained in an installed open plastic tube. Before the recording session, the tube is placed overnight in the cages that house the experimental animals to allow for habituation. The body temperature is maintained at ~ 27.7-28.8°C during the entire recording time. We recorded 3 experiments of 15 minutes for each mouse, mice are 8 weeks old and were treated with vehicle or G-2-MePE from Pl 5.

Night Activity Measurements

[00245] Spontaneous motor activity is measured by means of an infrared beam activated motion monitoring camera (Opto-Varimax-MiniA; Columbus Instruments, Columbus, Ohio). For each experiment, a mouse is placed in the chamber at least 3 hours before recording begins. Movement is monitored during the normal 12-hour dark cycle (seven hours to seven hours). One dark cycle per animal per time point is collected.

Results

[00246] To test whether G-2-MePE treatment will impact the development of the cardinal features of RTT disease, two-week-old mutant animals receive daily intraperitoneal injections over the course of their life. Measures of synaptic physiology, synaptic molecular composition, and cortical plasticity are then acquired as detailed below, along with health-related measures such as heart rate, levels of locomotor activity, and life.

Effects of G-2-MePE on the Synaptic Physiology of MeCP2 Mutant Mice

[00247] Recent studies have reported that neurons in various brain regions of MeCP2-/y mice exhibit a profound reduction in spontaneous activity (Chang et al., 2006;. Chao et al., 2007;. Dani et al., 2005; Nel- son et al. 2006) a phenotype that is rescued by overexpression of BDNF (Chang et al., 2006). Similarly, acute application of an IGF1 derivative has been shown to elevate excitatory-evoked post synaptic current (EPSC) amplitudes by 40% in rat hippocampal cultures (Ramsey et al., 2005, Xing et al., 2007). To test the effectiveness of G-2-MePE in rescue of the physiological phenotype MeCP2-/y, we acquired intracellular whole cell recordings in acute brain slices, measuring excitatory synaptic triggering (spontaneous EPSC amplitude and frequency) in layer 5 cortical neurons In this document EPSCs recorded from -/y animals are significantly reduced in amplitude compared to EPSCs measured from wild-type animals. The trend is partially reversed in EPSCs recorded from MeCP2-/y animals treated with G-2-MePE, which are significantly greater in amplitude than EPSCs from MeCP2-/y mice treated with vehicle. These differences are also seen when averaging in cells. Across these measurements, access resistance, leakage, and intrinsic cell excitability are also verified to be consistent across groups. Quantification of EPSC intervals also shows a slight increase in the interval between EPSC events (reduced EPSC frequency) between wild-type and MeCP2-/y animals (P = 0.04, Kolmogorov-Smirnov test). Our results thus indicate that the reduction of excitatory synaptic unit in cortical cells of MeCP2-/y mice and its partial recovery after G-2-MePE treatment are due, in part, to a change in EPSC amplitude as a consequence of a change in the resistance of the synapses that mediate excitatory transmission in this region.

G-2-MePE Treatment Stimulates Cortical Spine Maturation

[00248] We used Golgi staining to sparsely and distinctly label neurons and high resolution confocal imaging to measure dendritic spine density and morphology in the labeled cells restricting the analysis to layer 5 cortical neurons in the motor cortex sections from mice in the critical period (P28).

[00249] Although low magnification imaging distinctly delineates the extent of pyramidal cell dendrites we use higher magnifications to count synaptic contacts and determine the morphological class of each spine. We classify spines as either large and bulbous ("mushroom" M), short and thick ("corpulent", S), short and thin ("thin", T) or filopodia (F). Comparison of spine density by unit branch shows a trend of decreasing spine density in challenged neurons that is much improved in the treatment challenge.

[00250] Together, these results indicate the potential for deficits in the number and maturation state of dendritic contacts to support functional defects in excitatory transmission in a manner that can be treated after administration of G-2-MePE.

Ocular Dominance (OD) Plasticity in Adult Mice MeCP2 +/- is Reduced by G-2-MePE

[00251] Developmental changes in OD plasticity are controlled in part by activation of the IGF-1 pathway and administration of (1-3)IGF-1 can reduce OD plasticity in young wild-type mice (Tropea et al., 2006) . Therefore, we tested whether G-2-MePE treatment could stabilize the prolonged OD plasticity observed in adult MeCP2 mutants. Female MeCP2 +/- mice, aged P60 or older, are monocularly deprived for 4 days and concomitantly treated with G-2-MePE. Treatment with G-2-MePE reduces OD plasticity in adult MeCP2 +/- mice, indicating that, in fact, G-2-MePE can rapidly induce synapse stabilization or maturation.

Bradycardia in Mice MeCP2-/y is Treated by G-2-MePE

[00252] In addition to analyzing the effectiveness of G-2-MePE in alleviating neurophysiological symptoms, we sought to characterize its effects on the general health of the organism. Clinical and experimental evidence shows dysfunctions of the autonomic system, such as unstable respiratory rhythms and reduced baseline cardiac vagal tone in patients with Rett Syndrome (Julu et al., 2001). Lack of control of the feedback mechanisms that regulate blood pressure homeostasis through the sympathetic nervous system, eg decreased heart rate-induced hyperventilation, is common in patients with Rett Syndrome and can cause fatal cardiac arrhythmias (Acampa and Guideri, 2006; Julu et al., 2001).

[00253] The pathogenesis of cardiac dysautonomia, although not well understood, suggests that immature neuronal connections in the brain stem could be the cause. To analyze heart rhythm abnormalities in MeCP2-/y mice and the effect of G-2-Mepe treatment, we monitored the real-time cardiac pulse rate of unanesthetized wild-type and vehicle-treated MeCP2-/y animals. or G-2-MePE. Wild-type mice exhibited an even distribution of heart rate measurements centered near 750 beats per minute. In contrast, MeCP2-/y mice exhibit a more irregular heart rate, with a lower mean heart rate, the occurrence of which is significantly decreased after treatment with G-2-Mepe.

G-2-MePE Administration Improves Locomotor Activity and Life Expectancy

[00254] MeCP2-/y mice develop Rett-like symptoms beginning at 4-6 weeks of age, when they progressively become lethargic, develop gait ataxia, and die between 10 and 12 weeks of age (Chen et al., 2001) . Baseline locomotor activity is also recorded in mice after 6 weeks by counting nocturnal infrared beam crossing events within a fenced zone. MeCP2 knockout mice (KO) have significantly reduced levels of locomotor activity compared to wild-type (WT) mice, but treatment with G-2-MePE (KO-T) elevates these levels.

[00255] Finally, compared to MeCP2 KO litters, MeCP2-/y mice ratted with G-2-MePE also show a -50% increase in life expectancy (an increase in survival rate of 0.5 probability ).

[00256] We also measured the effect of G-2-MePE treatment on the size of soma neurons in the hippocampus. Mice are treated with G-2-MePE as described above for locomotor activity. The soma size in neurons in the CA3 region of the hippocampus is significantly impaired in MeCP2 KO animals compared to wild-type animals. Treatment with G-2-MePE increases mean soma size in KO animals, but has little or no effect on physical size in wild-type animals.

Example 11: Effect of Oral G-2-MePE on Survival in Rett Syndrome in Rats

[00257] Since Rett syndrome is a chronic, debilitating disorder involving the loss of motor skills, it is desirable to treat Rett syndrome using easily administered preparations. Unexpectedly beneficial pharmacokinetics of G-2-MePE compounds and related compounds (US Patent Nos. 7,041,314, 7,605,177, 7,714,070, 7,863,309 and US Patent Nos. 11/315,784 and 12/903,844).

[00258] Therefore, we administered G-2-MePE orally to MeCP2 deficient mice as described in US 2009/0074865. Briefly, an aqueous solution, a water-in-oil emulsion (microemulsion, coarse or liquid crystal emulsion), or a gel composition containing a pharmaceutically effective amount of G-2-MePE (20 or 80 mg/kg per animal ) is administered daily. In the MeCP2 deficient control animals, we administered only brine, and wild-type animals are used to obtain baseline data similar to the design studies described in Example 8, above.

[00259] In wild-type animals, survival is defined as being 100% at each time point. In animals deficient in MeCP2, survival is substantially reduced. However, after oral administration of G-2-MePE to MeCP2 deficient mice, survival is significantly increased.

Example 12: Effect of G-2MePE on Seizure Activity in Rett Syndrome in Mice

[00260] Since seizures are a prominent, dangerous and difficult to treat aspect of Rett Syndrome, we determined the effects of G-2MePE on seizure activity in MeCP2 deficient animals. G-2-MePE may be effective in treating seizure activity in animals with neurodegenerative disease, (US Patent number 7,714,020). Therefore, we performed experiments to determine whether G-2-MePE could also treat seizure activity in MeCP2-deficient mice.

[00261] Electroencephalographic recordings of wild-type mice and MeCP2-deficient mice treated with either brine or G-2 -MePE are obtained using the methods described in US Patent number 7,714,020.

[00262] We have found that G-2MePE can be effective in reducing both motor seizures and non-convulsive seizures.

Conclusions

[00263] Based on our in vivo and in vitro studies in MeCP2 deficient animals, we conclude that G-2-MePE may be an effective therapy for the treatment of humans with Rett syndrome. Furthermore, since G-2-MePE has an unexpectedly longer half-life than a naturally occurring compound ((1-3) IGF-1; glycyl-prolyl-glutamate or GPE) (figure 21), we can conclude that the use of G-2-MePE has distinct and substantial advantages over other pharmacological agents, including GPE.

[00264] For example, G-2-MePE is not degraded by gastrointestinal cells, being absorbed by gastrointestinal cells, and is active in the central nervous system after oral administration (Wen et al., US Application number 12/283,684; Patent US number 2009/0074865, US patent number 7,887,839, incorporated herein by reference in its entirety), Therefore, G-2MePE does not require intravenous, subcutaneous, intraventricular, or parenteral delivery. In fact, oral formulations comprising microemulsions, coarse dispersions, liquid crystal preparations, nanocapsules and hydrogels can be used in the manufacture of orally administered preparations, such as tablets, capsules and gels that can improve the neurological function and treat neurodegenerative diseases (US Patent number 7,887,839). The compounds of the present invention can be used in situations where the motor function of a patient is below what is necessary to swallow a tablet or capsule. There are several types of soluble gels for the oral administration of compounds, and these can be used to deliver a compound or composition of the present invention to a patient. Since G-2-MePE can be easily administered orally and orally effective in the treatment of neurodegenerative disorders, including Rett syndrome, we conclude that G-2-MePE can be convenient and beneficial for long-term therapy. in patients with Rett syndrome.

[00265] In addition, as Rett Syndrome shares important features with other autism spectrum disorders, the compounds of this invention may be useful to provide therapeutic benefit in animals with other ASD, and in humans with autism syndrome of Asperger's, Childhood Disintegrative Disorder and Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS).

Example 13: Treatment of ASD Shank3 Deficient Mouse Model

[00266] Shank-3 deficient mice are used in the study as a model of 22q l3 deletion syndrome associated with ASD.

[00267] The 22q l3 deletion syndrome has been associated with deletions or mutations in the Shank3 gene (Bonaglia et al., 2006). The shank3 gene codes for a master scaffolding protein constitute the structure at glutamatergic synapses (Boeckers et al., 2006). Shank3 is an essential part of the postsynaptic density (PSD) core and recruits several important functional elements for PSD and the synapse, including α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid components (AMPA), metabotropic glutamate (mGlu), and N-methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Recent studies exploring the 22q l3 clearance rate/shank3 mutations suggest that shank3 haploinsufficiency can cause a monogenic form of ASD with a frequency of 0.5% to 1% of ASD cases (Durand et al., 2007; Moessner et al., 2007; Gauthier et al., 2008).

[00268] The mouse model generation with interrupted expression of full-length shank3 has been previously described in the art (Bozdagi et al., Molecular Autism 2010, 1:15., P4). Briefly, Bruce4 C57BL/6 embryonic stem cells were used to generate a mouse strain that had loxP sites inserted before exon 4 and exon 9. The floxated allele was excised and one strain was maintained with a deletion of exons 4 -9, that is, a complete elimination of Shank3 ankyrin repeat domains. Heterozygous (+/-), wild type (+/+) and knockout (-/-) mice were produced with Mendelian frequencies from heterozygote-heterozygous crosses. A 50% reduction in the total length of Shank3 mRNA was confirmed in heterozygotes (qPCR), as well as a reduction in Shank3 protein expression by immunoblotting with Shank3 antibody N69/46).

[00269] The heterozygous mice generated by crossing wild-type and heterozygous mice are used in this example to better model the Shank3 haploinsufficiency responsible for the 22q l3 deletion syndrome.

Methods drug treatment

[00270] Wild-type, heterozygous Shank3-deficient mice, 1 to 3 months of age are divided into four treatment groups: placebo-treated wild-type, Shank3-deficient group treated with placebo and two groups treated with G Shank3 Deficient -2-MePE. Animals receive placebo (water) or G-2-MePE formulated in water and administered orally, b.i.d, for 14 days. G-2-MePE is administered in two doses: 15 or 60 mg/kg.

Methodology

[00271] A more detailed description of the methodology can be found in Bozdagi et al. (Molecular Autism 2010, 1:15).

Behavioral Analysis

[00272] Behavioral assessments are performed at various times, and include analysis of social interactions and social communication, ultrasonic, according to the methodology described by Bozdagi et al. Briefly, the social interactions between male and female in each group of treatment are evaluated. Males are housed in groups and individually tested in clean cages with clean sand. Each testing session lasts 5 minutes. Each of the mice is paired with an unfamiliar female C57BL/6J estrus female. A closed circuit television digital camera (Panasonic, Secaucus, New Jersey, US) is placed horizontally 30 cm from the room. An ultrasonic microphone (Advisorft UltraSoundGate CM 15 condenser microphone cap; Noticeft Bioacoustics, Berlin, Germany) is mounted 20 cm above the cage. Sampling frequency for the microphone is 250 kHz, and resolution is 16 bits. Although the equipment used cannot distinguish between the signals emitted by the male or his partner, the preponderance of signals during male-female interactions in mice is normally emitted by the male sex. The entire apparatus was contained in an ambient sound attenuation chamber (ENV-018V; Med Associates, St Albans, VT, USA), illuminated by a single 25 Watt red light. Videos of male subjects are further tagged by an uninformed investigator of the subject's genotype and treatment group on measurements of nose-to-nose sniffs, nose-to-anogenital sniffs, and sniffing of other regions of the body using the Observer Noldus software ( Noldus Information Technology, Leesburg, VA, USA). Ultrasonic vocalizations are identified manually by two highly trained investigators without knowledge of the genotype/treatment group information, and summary statistics are calculated using the Warningft package. The inter-rater reliability is 95%. Data are analyzed using unpaired Student's t-test.

[00273] Olfactory habituation/withdrawal test is conducted on male and female mice for each group. The methodology is as described above (Silverman et al. 2010, Yang et al. 2009 and Silverman et al. 2010). Non-social and social odors are presented on a series of cotton swabs inserted into the cage sequentially, each for 2 minutes, in the following order: water, water, water (distilled water), almond, almond, almond (1:100 extract dilution almond), banana, banana, banana (1:100 artificial banana flavor dilution); social 1, social 1, social 1 (swept from the bottom of an unfamiliar, matched sex B6 mouse housing cage); and social 2, social 2, social 2 (swept from the bottom and a second mouse housing cage 129/SvImJ, unknown, sex matched). One-way repeated ANOVA measures are performed within each treatment group for each set of habituation events and each withdrawal event, followed by a post hoc Tukey test.

Electrophysiology of Hippocampal Slices

[00274] Acute postmortem hippocampal slices (350 µm) are prepared from mice using a tissue cutter. Slices are kept and experiments are performed at 32°C. Slices are perfused with a Ringer's solution containing (in mM): NaCl, 125.0; C1, 2.5; MgSO4, 0.31; NaH2PO4, 0.0 1; NaHCO3, 26.2; CaCl2, 2.5; glucose, 11.0. Ringer's solution is bubbled with 95% O2/5% CO2 at 32°C during extracellular recordings (electrode solution: 3 M NaCl). Slices are maintained for 1 hour before establishing a baseline excitatory field post-synaptic potentials (fEPSPs) recorded from the radiatum stratum in the CA1 area, evoked by stimulation of Schaffer's collateral-commissural afferents (100 μs pulses every 30 seconds ) with bipolar tungsten electrodes placed in area CA3. The test stimulus intensity is adjusted to obtain fEPSPs with amplitudes that are half the maximum response. The initial EPSP slope (mV/MS) is determined from the mean waveform of four consecutive responses. Input-output (I/O) curves are generated by plotting the slope of fEPSP versus volley fiber amplitude in low Mg2+ (0.1 mM) solution. NMDA receptor-mediated and AMPA receptor-mediated I/O ratios are measured in the presence of ionotropic glutamate receptor antagonists: 2-amino-2-phosphonopantanoic acid APV (50 μM) and 6-cyano-7-nitroquinoxaline-2 ,3-dione CNQX (100 µM). Paired pulse responses are measured at interstimulus intervals from 10 to 200 ms, and are expressed as the ratio of the mean responses for the second stimulation pulse to the first stimulation pulse.

[00275] LTP is induced either by a high frequency stimulus (four trains of 100 Hz, stimulation of 1 sec. separated by 5 min.), or by theta-burst stimulation (TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms), or by a single 100 Hz stimulation, for control and genetically modified mice. To induce long-term depression (LTD), Schaffer collaterals are stimulated by a low frequency or low frequency stimulus (900 pulses at 1 Hz for 15 min.) to induce mGlu receptor-dependent LTD. Data are expressed as mean ± SD, and statistical analyzes are performed using analysis of variance (ANOVA) or Student's t test, with significance adjustment at an α level of 0.05.

Results behavioral

[00276] Cumulative duration of total social sniffs by male test mice is lower in the placebo-treated Shank3-deficient group than in the placebo-treated wild-type group. In addition, fewer ultrasonic vocalizations are emitted by the Shank3-deficient placebo-treated group than by wild-type controls during male-female social interactions.

[00277] Treatment with G-2-MePE in the two Shank-3 deficient groups results in a significant increase in the cumulative duration of total social sniffs compared to the Shank3 deficient group treated with placebo. Furthermore, the groups treated with G-2-MePE exhibit a greater number of ultrasonic vocalizations than the mutant group treated with placebo.

[00278] In the study of olfactory habituation/withdrawal, designed to confirm that mice are capable of detecting social pheromones, all four groups have normal levels of habituation (indicated by the decrease in time spent sniffing the sequence of the same three odors) , and expected weaning (indicated by increased time spent sniffing out different odors).

electrophysiology

[00279] Plotting the excitatory postsynaptic potential slope (fEPSP) versus stimulus intensity demonstrates a reduction in I/O curves in the Shank3-deficient group treated with placebo versus the control group. In the placebo-treated heterozygous group we also observed a decrease in AMPA receptor-mediated field potentials, reflected in a 50% decrease in the mean slope of I/O function compared to the wild-type control group. In contrast, when the IO ratio is analyzed in the presence of the competing antagonist AMPA/kainate receptor CNQX to measure synaptic NMDA receptor function, there is no difference between the wild-type and placebo treated heterozygous groups. These results indicate that there is a specific reduction in AMPA receptor-mediated basal transmission in heterozygous Shank3 mice.

[00280] Treatment with G-2-MePE in both heterozygous groups normalizes the field potentials mediated by AMPA receptors and causes an increase in the mean slope of the I/O function compared to the Shank3-deficient group treated with placebo.

[00281] The maintenance of LTP in the Shank3-deficient placebo-treated group is clearly impaired compared to the wild-type control. LTP TBS tests (10 bursts of four pulses at 100 Hz separated by 200 ms) also show a significant decrease in potentiation at 60 min. after TBS and in the Shank3-deficient placebo-treated group. In contrast to the altered synaptic plasticity seen with LTP, the long-term depression (LTD) was not significantly altered in the mutant group. Treatment with G-2-MePE increased long-term hippocampal potentiation (LTP) and its maintenance in both the Shank3-deficient group compared to the placebo-treated Shank3-deficient group.

Discussion

[00282] Weak social skills and repetitive behaviors are the common features and fundamental diagnostic measures of all forms of ASD. Delayed intellectual development and underdeveloped language skills are also a common feature present in all ASD, excluding Asperger syndrome.

[00283] The animal models described above have been accepted in the art as demonstrating symptoms similar to human clinical conditions. All of the mutant models discussed above (NLGN3, NLGN4, CADM 1, NRXN 1, FMR1, shank3) exhibit impaired social skills or increased social anxiety. Decreased excitatory transmission in the hippocampus has been identified in NRXN 1, shank3, MeCP2 and FMR1 mutant animal models. Currently, no polygenetic or multifactorial models of ASD have been described. The animal models described above, based on genetic abnormalities that are known to produce ASD in the human population, provide the best opportunity to test the effectiveness of ASD therapies.

[00284] Therefore, the effectiveness of G-2-MePE in animal models of ASD is reasonably predictive of its effectiveness in a human suffering from ASD.

Example 14: G-2-MePE Treatment Changes Neuron Morphology in an In Vitro Model of Rett Syndrome

[00285] To test the effects of G-2-MePE on neuronal morphology, an in vitro model of RTT described in Marchetto et al., A model for de-velopment and treatment of Rett syndrome using human induced pluripotent stem cells, Cell 143:527-539 (2010) (including supplementary information). The model uses induced pluripotent stem cells (iPSCs) generated from fibroblasts from human patients with RTT carrying different MeCP2 mutations.

Methods Cell Culture and Retrovirus Infection

[00286] RTT fibroblasts (carrying four distinct MeCP2 mutations) and control fibroblasts are generated from dermal biopsy explants. The shRNA against target MeCP2 gene is cloned into the Lenti-Lox3.7 lentivirus vector (as described in Marchetto et al.). Fibroblasts are infected with retroviral vectors (Reprogramming Sox2, Oct4, c-Myc and Kl4). Two days after infection, fibroblasts are seeded into mitotically inactivated mouse embryonic fibroblasts with hESC medium. After 2 weeks, iPSC colonies emerging from the bottom of the fibroblasts are manually harvested and transferred to feeder-free conditions on matrigel coated (BD) plates using mTeSR™ Embryonic Stem Cell Culture Medium (Stem Cell Technologies) and multiple passes manually. Gene expression profiles of the generated clones are measured using Affymetrix Gene Chip™ human genome arrays to confirm that the reprogramming is successful.

Neural Differentiation: NPCs and Mature Neurons

[00287] To obtain neural progenitor cells (NPCs), embryoid bodies (EBs) are formed by mechanical dissociation of cell groups and placement in low-adherence plates in hESC medium without FGF2 for 5-7 days. Afterwards, the EBs are placed on poly-ornithine/laminin coated plates in DMEM/F12 medium plus N2 medium (serum-free supplement for growth and expression of postmitotic cells). The resulting rosettes are collected after 7 days and dissociated with Accutase and seeded onto plates coated with NPC medium (DMEM/F12; 0.5X N2; 0.5X B27 and FGF2). Homogeneous populations of NPCs are achieved after 1-2 passes with Accutase in the same condition. To obtain mature neurons, floating EBs are treated with 1 µM or retinoic acid for 3 weeks (giving a total time to differentiation of 4 weeks). Mature EBs are dissociated with papain and DNAse for 1 hour at 37°C and placed on poly-ornithine/laminin coated plates in NPC medium without FGF2. Treatment with G-2-MePE RTT neuronal cultures are treated with G-2-MePE (1 nM-10 μM) for 1 week.

Immunocytochemistry and Quantification of Meuronal Morphology

[00288] Cells are fixed in 4% paraformic aldehyde and permeabilized with 0.5% Triton X-100 in PBS. Cells are then blocked with PBS containing 0.5% Triton X-100 and 5% donkey serum for 1 hour at room temperature. Fluorescent signals are detected using an inverted Zeiss microscope and images are processed with Photoshop CS3. The following primary antibodies are used: TRA-60, TRA-1-81 (1:00), Nanog and Lin28 (1:500), human nestin (1:100), Tuj-1 (1:500), Map2 (1:100); MeCP2 (1:1000); VGLUT1 (1:200), Psd95 (1:500), GFP (1:200), Soxl (1:250), Mushasi 1 (1:200) and me3H3 27 (1:500). The cell soma size is measured using a suitable software (eg ImageJ), after the identification of neurons using Syn :: EGFP™. The morphology of neuronal dendrites and spines are studied from an individual projection of z-stack optical sections and digitized in 0.5 μm increments that correlate with the evaluated resolution in the z-plane. Each optical section is the result of 3 scans at 500 lps followed by Kalman filtering. For synapse quantification, images are taken by a 1 μm z step using a Biorad radiance 2100™ confocal microscope. Synapse quantification is performed without knowledge regarding the genomic. Only VGLUTI points together with Map2-positive processes are counted. Statistical significance is tested using two-way ANOVA test and Bonferroni post-test.

Calcium Imaging

[00289] Neuronal networks derived from human iPSCs are infected with the lentivirus vector carrying the reporter construct Syn:DsRed. Cell cultures are washed twice with sterile Krebs HEPES buffer (KHB) and incubated with 2-5 µm Fluo-4AM™ (Molecular Probes/Invitrogen, Carlsbad, CA) in KHB for 40 minutes at room temperature. Excess dye is removed by washing twice with KHB, and an additional 20 minutes of incubation is performed to balance the intracellular dye concentration and allow for deesterification. Elapsed time (100X magnification) image sequences of 5,000 frames are acquired at 28 Hz with a region of 336 x 256 pixels, using a Hamamatsu ORCA-ER™, digital camera (Hamamatsu Photonics KK, Japan) with 488 nm filter (FITC) on an Olym-pus inverted fluorescence confocal microscope (Olympus Optical, Japan). Images are acquired with MetaMorph 7.7™ (MDS Analytical Technologies, Sunnyvale, CA). Images are subsequently processed using ImageJ™ and custom written routines in Matlab 7.2™ (Mathworks, Natick, MA).

electrophysiology

Whole cell patch clamp recordings are performed from cells co-cultured with astrocytes after 6 weeks of differentiation. The bath is constantly infused with fresh HEPES buffered brine (see complementary methods for recipe). The register micropipettes (36 MQ tip resistance) are filled with internal solution described in the supplementary materials. Recordings are performed using Axopatch 200B™ amplifier (Axon Instruments). Signals are filtered at 2 kHz and sampled at 5 kHz. The integral cell capacitance is fully compensated. The series resistance is decompensated, but monitored during the experiment by the amplitude of the capacitive current, in response to a 10 mV pulse. All registrations are carried out at room temperature and chemicals are purchased from Sigma. Frequency and amplitude of spontaneous postsynaptic currents are measured with the Mini Analysis Program™ software (Synaptosoft, Leonia, NJ). Statistical comparisons of WT and RTT groups are made using the non-parametric two-tailed Kolmogorov-Simirnov test with a significance criterion of p = 0.05. EPSCs are blocked by CNQX and DNQX (10-20 µm) and IPSPs are inhibited by bicuculline (20 µM).

Results

[00291] RTT iPSC-derived neurons are characterized by decreased number of glutamatergic synapses, reduced spine density, and smaller soma size. RTT neurons also show certain electrophysiological defects, that is, a significant decrease in the frequency and amplitude of spontaneous synaptic currents when compared to controls. RTT neurons show a lower frequency of intracellular calcium transitions.

[00292] We tested G-2-MePE in the above model to test whether any of the pathologies of the TTR phenotype could be attenuated.

[00293] Treating cell cultures with each drug concentration improves all morphological and physiological parameters of treated RTT cell cultures compared to untreated TTR controls. More specifically, a significant increase in the number of glutamatergic synapses is observed in cells treated with RTT L-2-MePE. All concentrations of G-2-MePE treatment increase the number of VGLUT1 points in RTT-derived neurons. G-2-MePE treatment normalizes the frequency and amplitude of spontaneous postsynaptic currents, as well as the transient frequency of calcium generated by synaptic activity of RTT neurons treated with G-2-MePE.

[00294] In the present in vitro model of human TTR, iPSCs derived from RTT patients and neurons differentiated from them are characterized by abnormalities in MeCP2 expression. As discussed in the detailed description of the invention above, the vast majority of RTT cases are associated with mutations in the MeCP2 gene. Therefore, the effectiveness of G-2-MePE in the present in vitro human TTR model is reasonably predictive of its effectiveness in a human subject suffering from TTR.

Example 15: Effects of G-2-MePE on Humans with Rett Syndrome Methods

[00295] Thirty individuals with Rett Syndrome are recruited. Individuals are female and aged between 16 and 29 years (mean = 12.1 SD = 4.4). All individuals have an IQ <60 and mutations in the MECP2 gene. Subjects also show spike ether activity in the EEG or an increase in the lower frequency ranges of the EEG detected by Fast Fourier Transform (FFT). Subjects are instructed that concomitant medications must be stable for at least six weeks prior to the study. Individuals receiving medication to treat signs of inattention are tested in the morning and instructed to take the medication in the afternoon. Individuals with QTc interval > 451 msec. are excluded.

The study is a double-blind, placebo-controlled, randomized parallel with three doses of placebo, 10 mg/kg three times daily orally G-2-MePE for five days, or 30 mg/kg three times per day orally with G-2-MePE.

[00297] Subjects are tested at baseline using the following instruments: The Rett Syndrome Natural Historry/Clinical Severity Scale, Aberrant Behavior Checklist Community Edition(ABC), Vinelands, Clinical Global Impression of Severity (CGI-S) and its caregivers completed the Caregiver Strain Questionnaire (CSQ).

[00298] Subjects are brought into the clinic in a hospital setting to allow initial reference recordings of EEG, ECG and respiratory rate continuously for 24 hours using polysomnography technology. Hand movements are also recorded using the Q-Sensor™. Derived EEG measurements include: spikes per unit of time in EEG, the total energy of the EEG, QTc, and heart rate variability (HRV) frequency ranges, and respiratory irregularities.

[00299] Adverse events are also recorded using standard safety measures and SMURF adverse event elicitation

[00300] Statistically, the effect of treatment with G-2-MePE is analyzed by performing a repeated analysis of covariance (ANCOVA) with the effect of treatment on changes in baseline counts.

Results

[00301] Treatment with G-2-MePE produces no more adverse effects than those present during treatment with placebo, with all adverse effects being of short duration and moderate severity. There are no reports of serious adverse events. There are no reports of cases of QTC increase. No effects are seen on respiratory rate or heart rate variability.

[00302] Treatment with G-2-MePE produces an overall significant reduction in spikes per unit time on the EEG. Treatment with 30 mg/kg three times a day orally of G-2-MePE decreases the peak activity compared to placebo. This dose of G-2-MePE also decreases the power of the delta band of the EEG compared to placebo.

[00303] Treatment with G-2-MePE also reduces total hand movements over a 24-hour period as counted using the Q-sensor™ device. This effect is significant for a dose of 30 mg T.I.D compared to placebo.

[00304] Treatment with G-2-MePE does not reduce the overall significant effect on the Rett Syndrome Natural Historry/Clinical Severity Score. However, 30 mg/kg of T.I.D. Oral G-2-MePE, compared to placebo, produces significant effects on the following subscales: "Nonverbal Communication at this visit by exam"; "Epilepsy/Seizures at this visit: and "Hand use".

Conclusions

[00305] Treatment with G-2-MePE produces significant improvements in central nervous system function in the present study. Despite the relatively short-term treatment, abnormalities in the brain's electrical activity are reduced, a clear sign of effectiveness. This effect is dose-dependent, seen after treatment with 30 mg/kg three times a day orally with G-2-MePE. These effects reflect the improvements in CNS function seen in the MECP2 knockout transgenic mouse model of Rett syndrome after administration of G-2-MePE.

[00306] Dose-dependent effects are also seen in the use of the hands, as assessed by an objective counting device and subjective assessment. This is of interest because unimportant hand pressure is characteristic for both the clinical phenotype of Rett Syndrome and is unique to this disorder. This measure primarily assesses eye contact. This raises the possibility that long-term treatment with G-2-MePE may improve social relationships in the population.

[00307] G-2-MePE is well tolerated in this population. No effect is seen either on standard measures or on specific areas of interest in the patient population, such as QT interval prolongation or apnea.

Example 16: Effects of G-2MePE on Humans with Autism Spectrum Disorders Methods

[00308] To determine whether G-2-MePE can treat the symptoms of ASD, we performed a study in humans with ASD. Twenty individuals with autism spectrum disorder are recruited. Subjects are male and aged between 16 and 65 years (mean = 18.1 SD = 3.4). All individuals have an IQ > 60 and a rigorous DSM-IV-TR diagnosis of Autism Disorder or Asperger's Disorder. Individuals also meet the criteria for an autism spectrum disorder per the ADI-R and ADOS-G documents and meet the proposed criteria for DSM-Vara and autism spectrum disorder. Subjects are instructed that concomitant medications must be stable for at least six weeks prior to the study. Individuals receiving medication to treat signs of inattention are tested in the morning and instructed to take the medication in the afternoon. Individuals who are best treated with atypical antipsychotic medications indicated for autism are excluded. Subjects are screened for known genetic disorders, including and those with Fragile X Syndrome or tuberous sclerosis are excluded. Individuals with uncontrolled epilepsy are excluded.

The study is a placebo-controlled, double-blind, three-phase crossover study. Individuals enter each stage of the mating in a random order. In the test phases, subjects receive either placebo, 10 mg/kg orally T.I.D G-2-MePE for five days or 30 mg/kg, three times a day orally of G-2-MePE. Each mating phase is separated by a 14-day washout period.

[00310] Subjects are tested at baseline, using the following instruments: Wechsler IQ, Abberant Behavior Checklist Community Edition (ABC), Vinelands, Yale-Brown Obsessive Compulsive Scale (YBOCS) compulsion subscale, Social Responsiveness Scale (SRS), Clinical Global Impressiono of Severity (CGI-S) and their caregivers complete the Caregiver Strain Questionnaire (CSQ).

[00311] Subjects receive two tasks - the Reading the Mind in the Eyes Test - Revised (RMET) and the Eye Tracking (ET) task as well as the Clinical Global Impression of Improvement (CGI-I). Tasks begin two hours after administration of placebo or any dose of LG2-MePE. The RMET is a computer-based task that assesses the ability to read eye emotions from subtle and affective facial expressions and is a widely used test of emotion recognition in patients with autism (2001). Importantly, RMET is able to detect improvement even with a single dose of a pharmacological agent (Guastella et al., 2010). Eye tracking issues are characteristic of patients with autism who spend less time looking into the eyes of photographs of human faces. Again, a single administration of a pharmacological intervention can improve eyes on human faces in autism (Andari et al., 2010).

[00312] Adverse events are also recorded with standard safety measures.

[00313] Statistically, the effect of treatment with G-2-MePE is analyzed by performing a repeated analysis of covariance (ANCOVA) with the effect of treatment on changes in baseline counts.

Results

[00314] Treatment with G-2-MePE does not produce more adverse effects than those present during treatment with placebo, with all adverse effects being of short duration and moderate severity. No serious adverse events are reported.

[00315] Treatment with G-2-MePE produces an overall significant improvement in the performance of the RMET test. Treatment with 30 mg/kg three times a day orally with G-2-MePE increases the percentage of correct responses in the RMET.

[00316] Treatment with G-2-MePE produces a significant improvement in the total time spent looking at the eye region in the ET test. CGI-I scores at the end of treatment periods show a significant difference. Positive treatment effects are correlated with baseline CSQ scores.

Conclusions

[00317] Treatment with G-2-MePE produces significant improvements in the Reading the Mind in the Eyes - Revised test and in the performance of an eye tracking task. This effect is dose-dependent, seen after treatment at 30 mg/kg , three times a day orally of G-2-MePE.

[00318] Improvement in these measures is a reflection of an improvement in the processing of social information processing. Social interaction deficits are a core diagnostic symptom for autism spectrum disorders, and this is therefore an important finding.

[00319] G-2-MePE also produces an overall improvement in function as indexed by Clinical Global Impression of Improvement. Free text annotation of the Case Report Forms of the study indicates that this effect is related to an improvement in social relationships. This implies that observed changes in RMET and ET task may have relevance to social activity in everyday life. G-2-MePE is well tolerated in this population.

Example 17: Animal Models for Determining the Effects of G-2-MePE on Autism Spectrum Disorders

[00320] The effects of G-2-MePE are further tested in the following genetic models of ASD: the heterozygous mouse Tbx1, the knockout mouse Cntnap2 and the knockout mouse Slc9a6. G-2-MePE is also tested in the fmrl knocked out mouse model of Fragile X Syndrome.

[00321] Tbxl. Mutations of the TBXI gene are associated with Autism Spectrum Disorders (Paylor et al., 2006). Transgenic Tbxl mice are selectively impaired in social interaction, ultrasonic vocalization, repetitive behaviors and working memory (Hiramoto et al., 2011).

[00322] Cntnap2. Two-thirds of patients with mutations of the contactin associated protein-like 2 (CNTNAP2) gene are given with an Autism Spectrum Disorder (Alarcon et al., 2008; Arking et al., 2008; Bakkaioglu et al., 2008; Strauss et al., 2006 ; Vernes et al., 2008). Cntnap2 knockout (KO) mice exhibit ASD-related phenotypes in social behavior, ultrasonic vocalization and repetitive behaviors (Penagarikano et al., 2011).

[00323] Slc9a6. This gene has been implicated in syndromic ASD and encodes the sodium hydrogen exchanger 6 (NHE6). Mutations in SLC9A6 are associated with intellectual disability (Gilfillan et al., 2008) and autistic behavior (Garbem et al., 2010). On S1c9a6 KO mice exhibit motor hyper-activity and cere-bellar dysfunction (Stromme et al., 2011).

[00324] Fmrl. Silencing of the FMRI gene produces Fragile X Syndrome, the phenotype of which includes autism; Two thirds of patients with Fragile X Syndrome meet screening criteria for an Autism Spectrum Disorder (Harris et al., 2008). Pediatric patients with Fragile X Syndrome also show lowered seizure threshold. The fmrl knockout mouse replicates much of the phenotype of Fragile X Syndrome, including juvenile seizure susceptibility (Van et al., 2004).

Methods

[00325] The animals in each of the models above are generated according to the methodology described in the cited literature. Wild-type equivalents are also obtained for each genetic template.

[00326] The animals in each model are divided into three groups (n = 10 and n = 20): wild-type mice treated with placebo, the mutant group treated with G-2-MePE and the mutant control group treated with placebo. Treatments are administered intraperitoneally: placebo (saline solution) or 20 mg/kg day of G-2-MePE.

[00327] Measures of key features of the ASD as presented in each model are taken according to the cited literature.

Results

[00328] Treatment with G-2-MePE significantly improves all measures associated with the ASD phenotype.

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[00329] The following references and all patents, patent applications and other publications cited herein are incorporated by reference.

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Claims (15)

1. Use of Glycyl-2-methyl-L-prolyl-L-glutamate (G-2-MePE) CHARACTERIZED by the fact that it is in the manufacture of a drug to treat a human having an autism spectrum disorder (ASD) . 2. Use according to claim 1, CHARACTERIZED by the fact that said ASD is selected from the group consisting of autism, fragile X chromosome syndrome, Rett syndrome (RTT), autism disorder, As- perger, Childhood Disintegrative Disorder and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), and Pathological Demand Avoidance (PDA). 3. Use, according to claim 1 or 2, CHARACTERIZED by the fact that said G-2-MePE is formulated for systemic, parenteral or direct administration to the brain. 4. Use, according to claim 1 or 2, CHARACTERIZED by the fact that said G-2-MePE is formulated for administration in a brain ventricle. 5. Use, according to claim 1 or 2, CHARACTERIZED by the fact that said G-2-MePE is formulated for administration through an intravenous, subcutaneous or oral route. 6. Use, according to any one of claims 1 to 5, CHARACTERIZED by the fact that said G-2-MePE is formulated for oral administration. 7. Use, according to any one of claims 1 to 6, CHARACTERIZED by the fact that said G-2-MePE is in a physiologically compatible aqueous solution, water-in-oil microemulsion, coarse water-in-oil emulsion, liquid crystal of water in oil, nanocapsules, or tablets. 8. Use according to any one of claims 1 to 7, CHARACTERIZED by the fact that said G-2-MePE is incorporated into a soluble gel in an aqueous solution. 9. Use, according to any one of claims 1 to 8, CHARACTERIZED by the fact that said G-2-MePE is formulated for administration in a mucosa of said human being. 10. Use, according to claim 8, CHARACTERIZED by the fact that said gel is formulated to be placed in the mouth of said human being. 11. Use of a compound of Formula 1 or Formula 2, CHARACTERIZED by the fact that it is in the manufacture of a drug to treat a human having an autism spectrum disorder (ASD):

where m is 0 or 1; n is 0 or 1; X is H or -NR6R7; Y is H, alkyl, -CO2R5, or -CONR6R7; Z is H, alkyl, -CO2R5 or -CONR6R7; R1 is H, alkyl, or aralkyl; R2, R3 and R4 are independently H or alkyl; each R5 is independently H, alkyl, or a fatty alcohol residue; each R6 and R7 is independently H, alkyl, or aralkyl, or -NR6R7 is pyrrolidine, piperidine, or morpholine; or a lactone formed when a compound where Y is -CO2(alkyl) and Z is -CO2H or where Y is -CO2H and Z is -CO2(alkyl) is lactonized; and the pharmaceutically acceptable salts thereof, provided that the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ or a salt thereof; wherein alkyl means a linear saturated hydrocarbyl group having from one to six carbon atoms, or a cyclic or branched saturated hydrocarbyl group having from three to six carbon atoms; and wherein aralkyl means a group of the formula -(CH2)1-2Ar, where Ar is a 5- or 6-membered heterocyclic or carbocyclic aromatic ring, optionally substituted with 1 to 3 substituents selected from Cl, Br, -OH, -O -alkyl, -CO2R8 (where R8 is H or alkyl), or -NR8R9, where R9 is H or alkyl. 12. Use, according to claim 11, CHARACTERIZED by the fact that: (a) the compounds are compounds of Formula 1; (b) m is 0; (c) n is 1; (d) X, Y, R1, R2, R3, R4 or R5, or any combination thereof, is not hydrogen; (e) X is -NR6R7; and (f) Y is -CO2R5 or -CO2NR6R7; and (g) Z is -CO2R5 or -CO2NR6R7. 13. Use, according to any one of claims 1 to 12, CHARACTERIZED by the fact that said drug further comprises a second therapeutic agent selected from a group consisting of: insulin-like growth factor I (IGF- I), insulin-like growth factor II (IGF-II), glycyl-prolyl-glutamate (GPE), transforming growth factor-β1, activin, growth hormone, neural growth factor, brain-derived neurotrophic factor (BDNF ), growth hormone binding protein, IGF binding proteins (especially IGFBP-3), basic fibroblast growth factor, acidic fibroblast growth factor, the hst/Kfgk gene product, FGF-3 , FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor, int-2, homologous factor 1 to fibroblast growth factor (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, cell activating factor gl ials, FGF-10 and FGF-16, ciliary neurotrophic factor, brain-derived growth factor, neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial cell line-derived neurotrophic factor, neurotrophic factor activity-dependent, cytokine leukemia inhibitory factor, oncostatin M, interleukin, α-, β-, Y- or consensus interferon, TNF-α, clomethiazole; kynurenic acid, Semax, tacrolimus, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, andrenocorticotropin analogue (4-9) [ORG 2766], dizolcipine (MK-801), selegiline ; mematin (Namenda) NPS1506, GV1505260, MK-801, GV150526; AMPA antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164, fenobam, a selective serotonin reuptake inhibitor such as fluoxetine, or an atypical antipsychotic such as risperidone. 14. Use according to claim 1 or 11 CHARACTERIZED by the fact that the treatment of said ASD results in an improvement of: (a) impairment in social interaction, (b) impairment in communication, (c) restricted interests and behaviors and repetitive, (d) atypical eating, (e) deficiency in the use of multiple non-verbal behaviors to regulate social interaction; (f) inability to develop developmentally appropriate peer relationships, (g) lack of spontaneous seeking to share pleasure, interests or achievements, (h) lack of social or emotional reciprocity, (i) delay in or total lack of spoken language development, (j) marked impairment in the ability to initiate or maintain a conversation, (k) stereotyped and repetitive use of language, (l) lack of spontaneous make-believe play, (m) restricted patterns , repetitive and stereotyped behavior, (n) preoccupation with one or more interest(s) considered abnormal in intensity, (o) inflexible adherence to routines or rituals, (p) repetitive motor mannerisms, or (r) ) persistent focus on parts of objects. 15. Use according to claim 1 or 11, CHARACTERIZED by the fact that said ASD is Rett Syndrome.

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